Antibodies to Canine Interleukin-4 Receptor Alpha

ABSTRACT

The present invention discloses antibodies and blocking antibodies to canine IL-4 receptor alpha that have specific sequences and a high binding affinity for canine IL-4 receptor α. The present invention also discloses the use of the antibodies of the present invention in the treatment of atopic dermatitis in dogs. The present invention further discloses unique epitopes that bind to the antibodies to canine IL-4 receptor alpha.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) of provisionalapplications U.S. Ser. Nos. 62/142,108 filed Apr. 2, 2015; 62/269,486filed Dec. 18, 2015, and 62/310,250 filed Mar. 18, 2016, the contents ofall of which are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to antibodies to canine IL-4 receptoralpha that have specific sequences, a high binding affinity for canineIL-4 receptor alpha, including some that can block the binding of canineIL-4 to canine IL-4 receptor alpha. The present invention furtherrelates to unique epitopes that bind to the antibodies to canine IL-4receptor alpha. The present invention also relates to use of theantibodies and the epitopes of the present invention in the treatment ofatopic dermatis in dogs.

BACKGROUND OF THE INVENTION

The immune system comprises a network of resident and recirculatingspecialized cells that function collaboratively to protect the hostagainst infectious diseases and cancer. The ability of the immune systemto perform this function depends to a large extent on the biologicalactivities of a group of proteins secreted by leukocytes andcollectively referred to as interleukins. Among the well-studiedinterleukins are two important molecules identified as interleukin-4(IL-4) and interleukin-13 (IL-13). IL-4 and IL-13 are two closelyrelated proteins that can be secreted by many cell types including CD4⁺Th2 cells, natural killer T cells (NKT), macrophages, mast cells, andbasophils. IL-4 and IL-13 display many overlapping functions and arecritical to the development of T cell-dependent humoral immuneresponses. Despite their similarities in overall structure, cell sourcesand biological functions, each of these cytokines mediates certainspecialized functions, which has stimulated considerable research aimedat identifying the receptors and the downstream signaling pathwaysthrough which these interleukins mediate both their common and uniquebiological activities.

It is now known that IL-4 binds with high affinity to two receptorsi.e., type-I and type-II IL-4 receptors. The type I IL-4 receptorconsists of the IL-4 receptor α chain and the common γ C chain, which isalso part of the receptor for several other interleukins including IL-2,IL-7, IL-9, and IL-15. The Type II IL-4 receptor consists of the IL-4receptor α chain and the IL-13 receptor α1 chain. On other hand, IL-13binds to the type-II IL-4 receptor, and to a unique receptor designatedIL-13 receptor α2. The binding of IL-13 to the IL-13 receptor α2 doesnot transduce a signal and this receptor is also secreted in a solubleform. Accordingly the IL-13 receptor α2 has often been referred to as adecoy receptor.

The genes encoding the IL-4 protein from various species have beencloned and expressed in bacterial and mammalian cells. For example, thecDNA encoding human IL-4 shows that the mature human IL-4 is a secretedpolypeptide of 129 amino acids with a predicted molecular weight of 15Kd [Yokota et al., Proc Natl Acad Sci USA. 83(16): 5894-5898 (1986)].The cDNA encoding the canine IL-4 protein has also been identified andshown to encode a 132 amino acid polypeptide that shares 40% identitywith human IL-4 [van der Kaaij et al., Immunogenetics 49:142-143(1999)]. The gene encoding human IL-13 has been cloned and expressed ina variety of host systems [Minty et al., Nature 362:248-50 (1993)]. AcDNA encoding human IL-13 shows that the mature IL-13 is a secretedpolypeptide with a 12.4 Kd apparent molecular weight. A cDNA encodingcanine IL-13 also has been identified [Yang et al., J. Interferon andCytokine Research 20:779-785 (2000)]. The predicted canine IL-13 maturepolypeptide consists of 111 amino acids and shares 61.8% identity withhuman IL-13.

The genes encoding the human and mouse IL-4 receptor α chains have beencloned and expressed in a variety of host systems. For example, the cDNAencoding the human IL-4 receptor α chain has been described by Galizziet al., [International Immunology 2(7):669-675 (1990)] and the cDNAencoding the murine IL-4 receptor α chain has been described by Mosleyet al., [Cell, 59(2):335-348 (1989)]. The cDNA for human IL-4 receptor αchain encodes for 825 amino acid residues including a 24 amino acidresidue signal sequence. Although the murine protein is 15 amino acidresidues shorter than the human receptor, both proteins are closelyrelated with an overall sequence identity of 50% at the amino acidlevel.

Genes encoding equine, canine, and feline IL-4 receptor α chains havealso been disclosed [see, U.S. Pat. No. 7,208,579 B2]. In addition, acDNA predicted to be corresponding to one isoform of canine IL-4receptor α can be found in Genbank database (SEQ ID NO: 1). The presentinvention therefore undertook to determine the IL-4 receptor α chaincDNA and to definitively determine its encoded polypeptide sequence.

Although IL-4 and IL-13 are critical cytokines for the development ofTh2 immune responses that are required for protection againstextracellular pathogens (e.g., tissue or lumen dwelling parasites), bothcytokines have been implicated in the pathogenesis of a variety ofallergic diseases in humans and animals, including asthma and atopicdermatitis. Asthma is a common respiratory disease in humans. Thedisease is characterized by lung inflammation, hyper-responsiveness ofbronchial airways to external stimuli, and structural modifications ofthe bronchial wall tissues. The pathophysiology of allergic asthma hasbeen reviewed by Vatrella et al., [Journal of Asthma and Allergy7:123-130 (2014)]. Asthma is sustained by CD4⁺ Th2 cells which producelarge amounts of IL-4 and IL-13 and orchestrate the immune inflammatoryresponse in the allergic airways. Recent progress in understanding theasthmatic response highlights the important roles played by both IL-4and IL-13 in the disease pathogenesis. For example, both cytokinesstimulate immunoglobulin isotype switch in B cells from IgM to IgE, andthis allergen-specific IgE contribute to mast cell degranulation andrelease of inflammatory mediators in the airways. In addition, both IL-4and IL-13 increase bronchial smooth muscle contraction and stimulateairway recruitment of eosinophils which can also degranulate in responseto crosslinking of allergen-bound IgE to its receptor on eosinophils. Inaddition, IL-13 also stimulates mucus secretion and promotes airwayremodeling by stimulating goblet cell hyperplasia, deposition ofcollagen, and proliferation of airway smooth muscle cells. Thus it isnow clear that IL-4 and IL-13 are intimately involved in thepathological changes that lead to expression of asthmatic episodesincluding bronchial constriction and increased airway hyperactivity.

Atopic dermatitis (AD) is a relapsing pruritic inflammatory skin diseasethat is characterized by immune system dysregulation and epidermalbarrier abnormalities. The pathological and immunological attributes ofAD have been the subject of extensive investigations [reviewed in Rahmanet al. Inflammation & Allergy-drug target 10:486-496 (2011) and Harskampet al., Seminar in Cutaneous Medicine and Surgery 32:132-139 (2013)]. ADis the most common skin disease in man affecting 2-10% of the adultpopulation in the United States and about 25% of children worldwide. Inman, AD skin lesions are characterized by infiltrations with Th2 cells,eosinophils, mast cells and dendritic cells. In the acute phase of AD,these lesions display a predominant expression of Th2-type cytokinesincluding IL-4 and IL-13. AD is also characterized by elevatedcirculating levels of IgE and is positively correlated with IL-4 andIL-13 expression in CD4⁺ Th2 cells in the skin. Although AD has beenclassified as a Th2 disease, other T cell subsets such as Th1, Th22 andTh17 might also contribute to disease pathogenesis. Despite theincreasing incidence of AD worldwide, treatment options available topatients whose symptoms are not adequately controlled by topical agentsare limited to oral corticosteroids, oral cyclosporine and narrow bandUVB phototherapy. These therapies are not always effective and their useis associated with a variety of safety effects. Recently, monoclonalantibodies specific to human IL-4 R_(a) have been developed and some ofthese antibodies have been tested extensively for their therapeuticutilities in man for treatment of atopic dermatitis [see, e.g,US20150017176 A1].

AD is also a common disease in companion animals, especially dogs, whereits prevalence has been estimated to be approximately 10-15% of thecanine population. The pathogenesis of AD in dogs and cats [reviewed inNuttall et al., Veterinary Records 172(8):201-207 (2013)] bearssignificant similarities to that of AD in man including skininfiltration by a variety of immune cells and CD4⁺ Th2 polarizedcytokine milieu including preponderance of IL-4 and IL-13 cytokines. Asin humans, current therapies for atopic dermatitis in dogs and cats relyon palliative therapy such as shampoos and moisturizers or symptomatictherapy via the use of oral or systemic corticosteroids and oralcyclosporine. As with human AD, these therapies do not address theunderlying mechanism of disease and have significant safety and efficacyissues. Thus, there is an unmet medical need for a safe and effectivetreatment option for AD in companion animals. Such treatment shouldpreferably interfere with the underlying mechanism of disease.

The citation of any reference herein should not be construed as anadmission that such reference is available as “prior art” to the instantapplication.

SUMMARY OF THE INVENTION

The present invention relates to anti-canine interleukin-4 receptoralpha (IL-4R_(a)) antibodies that have a high binding affinity forcanine IL-4R_(a). In more particular embodiments, the anti-canineinterleukin-4 receptor alpha (IL-4R_(a)) antibodies also have theability to block the binding of canine IL-4 and canine IL-13 to thetype-I or type II IL-4 receptors and subsequently inhibit the signalingfrom both canine IL-4 and IL-13. In particular embodiments suchanti-canine IL-4R_(a) antibodies are murine anti-canine IL-4R_(a)antibodies. In more particular embodiments the anti-canine IL-4R_(a)antibodies have a high binding affinity to canine IL-4R_(a), as well ashave the ability to block the binding of canine IL-4 and canine IL-13 tothe type-I and type II IL-4 receptors.

Moreover, the present invention relates to the complementary determiningregions (CDRs) comprised by these antibodies and the combination ofthese CDRs (e.g., obtained from murine anti-canine IL-4R_(a) antibodies)into canine frames to form caninized anti-canine IL-4R_(a) antibodies.The present invention also relates to use of such antibodies in thetreatment of conditions such as atopic dermatitis and/or other adverseconditions due to the downstream effects of the signaling from thebinding of canine IL-4 and/or canine IL-13 to the type-I and/or type IIIL-4 receptors.

Accordingly, the present invention provides unique sets of CDRs fromfourteen (14) exemplified murine anti-canine IL-4R_(a) antibodies. The14 exemplified murine anti-canine IL-4R_(a) antibodies have unique setsof CDRs, i.e., three light chain CDRs: CDR light 1 (CDRL1), CDR light 2(CDRL2), and CDR light 3 (CDRL3) and three heavy chain CDRs CDR heavy 1(CDRH1), CDR heavy 2 (CDRH2) and CDR heavy 3 (CDRH3). As detailed below,there is substantial sequence homology within each group of CDRs, andeven some redundancy e.g., see, the set of CDRL1s below. Therefore, thepresent invention not only provides the amino acid sequences of the sixCDRs from the 14 exemplified murine anti-canine IL-4R_(a) antibodies,but further provides conservatively modified variants of these CDRs, aswell as variants that comprise (e.g., share) the same canonicalstructure and/or bind to one or more (e.g., 1 to 4, or more) amino acidresidues of canine IL-4R_(a) that are comprised by an epitope of canineIL-4R_(a).

Therefore, the present invention provides an antibody or antigen bindingfragment thereof that binds IL-4R_(a) with specificity comprising alight chain complementary determining region 1 (VL CDR1) that comprisesthe amino acid sequence of SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49,SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO:54, SEQ ID NO: 55, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131,and/or a light chain complementary determining region 2 (VL CDR2)comprising the amino acid sequence of SEQ ID NO: 56, SEQ ID NO: 57, SEQID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62,SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 132, SEQ ID NO: 133, or SEQ IDNO: 134, and/or a light chain complementary determining region 3 (VLCDR3) comprising the amino acid sequence of SEQ ID NO: 65, SEQ ID NO:66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ IDNO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 135, SEQ ID NO: 136,SEQ ID NO: 137, SEQ ID NO: 138, or SEQ ID NO: 139, and/or a heavy chaincomplementary determining region 1 (VH CDR1) in which the CDRH1comprises the amino acid sequence of SEQ ID NO: 74, SEQ ID NO: 75, SEQID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80,SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO:142, or SEQ ID NO: 143, and/or a heavy chain complementary determiningregion 2 (VH CDR2) comprising the amino acid sequence of SEQ ID NO: 83,SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO:88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 144, SEQ IDNO: 145, SEQ ID NO: 146, SEQ ID NO: 147, or SEQ ID NO: 148 and/or aheavy chain complementary determining region 3 (VH CDR3) comprising theamino acid sequence of SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99,SEQ ID NO: 100, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ IDNO: 152, or SEQ ID NO: 153. In particular embodiments the antibody is amammalian antibody. In more particular embodiments the antibody is acaninized antibody.

Accordingly, a caninized antibody of the present invention or antigenbinding fragment thereof comprises one or more of the heavy chaincomplementary determining region 1 (VH CDR1) with an amino acid sequenceof SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ IDNO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, or SEQ ID NO: 143. Inanother embodiment, the heavy chain complementary determining region 2(VH CDR2) comprises an amino acid sequence of SEQ ID NO: 83, SEQ ID NO:84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ IDNO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ ID NO: 144, SEQ ID NO: 145,SEQ ID NO: 146, SEQ ID NO: 147, or SEQ ID NO: 148. In still anotherembodiment the heavy chain complementary determining region 3 (VH CDR3)comprises an amino acid sequence of SEQ ID NO: 92, SEQ ID NO: 93, SEQ IDNO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQID NO: 99, SEQ ID NO: 100, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO:151, SEQ ID NO: 152, or SEQ ID NO: 153. In a particular embodiment ofthis type, the caninized antibody or antigen binding fragment comprisesboth a VH CDR1 comprising an amino acid sequence of SEQ ID NO: 74, SEQID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79,SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 140, SEQ ID NO:141, SEQ ID NO: 142, or SEQ ID NO: 143 and a VH CDR2 comprising an aminoacid sequence of SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO:86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ IDNO: 91, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147,or SEQ ID NO: 148. In another such embodiment, the caninized antibody orantigen binding fragment comprises both a VH CDR1 comprising an aminoacid sequence of SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO:77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ IDNO: 82, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 142, or SEQ ID NO:143, and a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 92,SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO: 100, SEQ ID NO: 149, SEQID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, or SEQ ID NO: 153. In yetanother such embodiment, the caninized antibody or antigen bindingfragment comprises both a VH CDR2 comprising an amino acid sequence ofSEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO:87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, SEQ ID NO: 91, SEQ IDNO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQ ID NO: 147, or SEQ ID NO:148 and a VH CDR3 comprising an amino acid sequence of SEQ ID NO: 92,SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO:97, SEQ ID NO: 98, SEQ ID NO: 99, or SEQ ID NO: 100, SEQ ID NO: 149, SEQID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152, or SEQ ID NO: 153. In stillanother such embodiment, the caninized antibody or antigen bindingfragment comprises a VH CDR1 comprising an amino acid sequence of SEQ IDNO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 140,SEQ ID NO: 141, SEQ ID NO: 142, or SEQ ID NO: 143, a VH CDR2 comprisingan amino acid sequence of SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85,SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO:90, SEQ ID NO: 91, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, SEQID NO: 147, or SEQ ID NO: 148 and a VH CDR3 comprising an amino acidsequence of SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95,SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, or SEQ IDNO: 100, SEQ ID NO: 149, SEQ ID NO: 150, SEQ ID NO: 151, SEQ ID NO: 152,or SEQ ID NO: 153.

In particular embodiments, the caninized antibody or antigen bindingfragment also comprises a light chain complementary determining region 1(VL CDR1) comprising an amino acid sequence of SEQ ID NO: 47, SEQ ID NO:48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ IDNO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 129, SEQ ID NO: 130, orSEQ ID NO: 131. In related embodiments the light chain complementarydetermining region 2 (VL CDR2) comprises an amino acid sequence of SEQID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60,SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO:132, SEQ ID NO: 133, or SEQ ID NO: 134. In still another embodiment thelight chain complementary determining region 3 (VL CDR3) comprises anamino acid sequence of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72,SEQ ID NO: 73, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ IDNO: 138, or SEQ ID NO: 139. In a particular embodiment of this type, thecaninized antibody or antigen binding fragment comprises both a VL CDR1comprising an amino acid sequence of SEQ ID NO: 47, SEQ ID NO: 48, SEQID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53,SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ IDNO: 131 and a VL CDR2 comprising an amino acid sequence of SEQ ID NO:56, SEQ ID NO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ IDNO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 132, SEQID NO: 133, or SEQ ID NO: 134.

In other such embodiments, the caninized antibody or antigen bindingfragment comprises both a VL CDR1 comprising an amino acid sequence ofSEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ IDNO: 129, SEQ ID NO: 130, or SEQ ID NO: 131 and a VL CDR3 comprising anamino acid sequence of SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72,SEQ ID NO: 73, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, SEQ IDNO: 138, or SEQ ID NO: 139. In yet another such embodiments, thecaninized antibody or antigen binding fragment comprises both a VL CDR2comprising an amino acid sequence of SEQ ID NO: 56, SEQ ID NO: 57, SEQID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62,SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 132, SEQ ID NO: 133, or SEQ IDNO: 134 and a VL CDR3 comprising an amino acid sequence of SEQ ID NO:65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ IDNO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 135, SEQID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, or SEQ ID NO: 139.

In still other such embodiments, the caninized antibody or antigenbinding fragment comprises a VL CDR1 comprising an amino acid sequenceof SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ IDNO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131, a VL CDR2 comprising anamino acid sequence of SEQ ID NO: 56, SEQ ID NO: 57, SEQ ID NO: 58, SEQID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63,SEQ ID NO: 64, SEQ ID NO: 132, SEQ ID NO: 133, or SEQ ID NO: 134, and aVL CDR3 comprising an amino acid sequence of SEQ ID NO: 65, SEQ ID NO:66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ IDNO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 135, SEQ ID NO: 136,SEQ ID NO: 137, SEQ ID NO: 138, or SEQ ID NO: 139.

In particular embodiments the caninized anti-canine IL-4R_(a) antibodycomprises complementary determining regions (CDRs) in which the CDRshave canonical structures of: H1-1, H2-3A, and H3-12, respectively forCDR1, CDR2, and CDR3 of the heavy chain, i.e., CDR1 of the heavy chainhas the canonical structure class 1, CDR2 of the heavy chain has thecanonical structure class 3A, and CDR3 of the heavy chain has thecanonical structure class 12. In even more particular embodiments, theCDRs for the corresponding light chains have canonical structures of:L1-1, L2-1, and L3-1, respectively for CDR1, CDR2, and CDR3 of the lightchain. In other embodiments the caninized anti-canine IL-4R_(a) antibodycomprises complementary determining regions (CDRs) in which the CDRshave canonical structures of: H1-1, H2-2A, and H3-7, respectively forCDR1, CDR2, and CDR3 of the heavy chain. In even more particularembodiments of this type, the CDRs for the corresponding light chainshave canonical structures of: L1-2A, L2-1, and L3-1, respectively forCDR1, CDR2, and CDR3 of the light chain. In still other embodiments thecaninized anti-canine IL-4R_(a) antibody further comprises complementarydetermining regions (CDRs) in which the CDRs have canonical structuresof: H1-1, H2-2B, and H3-15, respectively for CDR1, CDR2, and CDR3 of theheavy chain. In even more particular embodiments of this type, the CDRsfor the corresponding light chains have canonical structures of: L1-4,L2-1, and L3-1, respectively for CDR1, CDR2, and CDR3 of the lightchain. In yet other embodiments the caninized anti-canine IL-4R_(a)antibody further comprises complementary determining regions (CDRs) inwhich the CDRs have canonical structures of: H1-1, H2-1, and H3-15,respectively for CDR1, CDR2, and CDR3 of the heavy chain. In even moreparticular embodiments of this type, the CDRs for the correspondinglight chains have canonical structures of: L1-3, L2-1, and L3-1,respectively for CDR1, CDR2, and CDR3 of the light chain. In still otherembodiments the caninized anti-canine IL-4R_(a) antibody furthercomprises complementary determining regions (CDRs) in which the CDRshave canonical structures of: H1-1, H2-2B, and H3-6, respectively forCDR1, CDR2, and CDR3 of the heavy chain. In even more particularembodiments of this type, the CDRs for the corresponding light chainshave canonical structures of: L1-2A, L2-1, and L3-1, respectively forCDR1, CDR2, and CDR3 of the light chain.

In yet other embodiments the caninized anti-canine IL-4R_(a) antibodyfurther comprises complementary determining regions (CDRs) in which theCDRs have canonical structures of: H1-1, H2-2B, and H3-4, respectivelyfor CDR1, CDR2, and CDR3 of the heavy chain. In even more particularembodiments of this type, the CDRs for the corresponding light chainshave canonical structures of: L1-6, L2-1, and L3-1, respectively forCDR1, CDR2, and CDR3 of the light chain. In still other embodiments thecaninized anti-canine IL-4R_(a) antibody further comprises complementarydetermining regions (CDRs) in which the CDRs have canonical structuresof: H1-1, H2-1, and H3-13, respectively for CDR1, CDR2, and CDR3 of theheavy chain. In even more particular embodiments of this type, the CDRsfor the corresponding light chains have canonical structures of: L1-1,L2-1, and L3-1, respectively for CDR1, CDR2, and CDR3 of the lightchain. In yet other embodiments the caninized anti-canine IL-4R_(a)antibody further comprises complementary determining regions (CDRs) inwhich the CDRs have canonical structures of: H1-1, H2-2A, and H3-6,respectively for CDR1, CDR2, and CDR3 of the heavy chain. In even moreparticular embodiments of this type, the CDRs for the correspondinglight chains have canonical structures of: L1-2A, L2-1, and L3-1,respectively for CDR1, CDR2, and CDR3 of the light chain.

In still other embodiments the caninized anti-canine IL-4R_(a) antibodyfurther comprises complementary determining regions (CDRs) in which theCDRs have canonical structures of: H1-1, H2-3A, and H3-15 oralternatively H3-13, respectively for CDR1, CDR2, and CDR3 of the heavychain. In even more particular embodiments of this type, the CDRs forthe corresponding light chains have canonical structures of: L1-6, L2-1,and L3-1, respectively for CDR1, CDR2, and CDR3 of the light chain. Inyet other embodiments the caninized anti-canine IL-4R_(a) antibodyfurther comprises complementary determining regions (CDRs) in which theCDRs have canonical structures of: H1-1, H2-2A, and H3-10, respectivelyfor CDR1, CDR2, and CDR3 of the heavy chain. In even more particularembodiments of this type, the CDRs for the corresponding light chainshave canonical structures of: L1-6, L2-1, and L3-1, respectively forCDR1, CDR2, and CDR3 of the light chain. In still other embodiments thecaninized anti-canine IL-4R_(a) antibody further comprises complementarydetermining regions (CDRs) in which the CDRs have canonical structuresof: H1-1, H2-3A, and H3-9, respectively for CDR1, CDR2, and CDR3 of theheavy chain. In even more particular embodiments of this type, the CDRsfor the corresponding light chains have canonical structures of: L1-3,L2-1, and L3-3, respectively for CDR1, CDR2, and CDR3 of the lightchain.

The present invention also provides an isolated caninized antibody orantigen binding fragment thereof that specifically binds IL-4R_(a)comprising a canine IgG heavy chain and a canine kappa or lambda lightchain. In particular embodiments of this type, the canine kappa orlambda light chain that comprises three light chain complementarydetermining regions (CDRs): CDR light 1 (CDRL1), CDR light 2 (CDRL2),and CDR light 3 (CDRL3); and the canine IgG heavy chain comprises threeheavy chain CDRs: CDR heavy 1 (CDRH1), CDR heavy 2 (CDRH2) and CDR heavy3 (CDRH3) is obtained from the murine anti-canine IL-4R_(a) antibodies.Particular embodiments of the caninized antibodies and antigen bindingfragments thereof of the present invention bind canine IL-4R_(a) and/orblock the binding of canine IL-4R_(a) to canine IL-4.

In specific embodiments, the present invention provides an isolatedmammalian antibody or antigen binding fragment thereof that binds canineinterleukin-4 receptor alpha (IL-4R_(a)) with specificity comprisingthree light chain complementary determining regions (CDRs): CDR light 1(CDRL1), CDR light 2 (CDRL2), and CDR light 3 (CDRL3); and three heavychain CDRs: CDR heavy 1 (CDRH1), CDR heavy 2 (CDRH2) and CDR heavy 3(CDRH3). In certain embodiments the CDRL1 comprises the amino acidsequence of SEQ ID NO: 47, a variant of SEQ ID NO: 47, a conservativelymodified variant of SEQ ID NO: 47, a variant of SEQ ID NO: 47 thatcomprises the canonical structure class of 1, SEQ ID NO: 48, a variantof SEQ ID NO: 48, a conservatively modified variant of SEQ ID NO: 48, avariant of SEQ ID NO: 48 that comprises the canonical structure class of2A, SEQ ID NO: 49, a variant of SEQ ID NO: 49, a conservatively modifiedvariant of SEQ ID NO: 49, a variant of SEQ ID NO: 49 that comprises thecanonical structure class of 4, SEQ ID NO: 50, a variant of SEQ ID NO:50, a conservatively modified variant of SEQ ID NO: 50, a variant of SEQID NO: 50 that comprises the canonical structure class of 3, SEQ ID NO:51, a variant of SEQ ID NO: 51, a conservatively modified variant of SEQID NO: 51, a variant of SEQ ID NO: 51 that comprises the canonicalstructure class of 3, SEQ ID NO: 52, a variant of SEQ ID NO: 52, aconservatively modified variant of SEQ ID NO: 52, a variant of SEQ IDNO: 52 that comprises the canonical structure class of 2A, SEQ ID NO:53, a variant of SEQ ID NO: 53, a conservatively modified variant of SEQID NO: 53, a variant of SEQ ID NO: 53 that comprises the canonicalstructure class of 6, SEQ ID NO: 54, a variant of SEQ ID NO: 54, aconservatively modified variant of SEQ ID NO: 54, a variant of SEQ IDNO: 54 that comprises the canonical structure class of 1, SEQ ID NO: 55,a variant of SEQ ID NO: 55, a conservatively modified variant of SEQ IDNO: 55, a variant of SEQ ID NO: 55 that comprises the canonicalstructure class of 2A, SEQ ID NO: 129, a variant of SEQ ID NO: 129, aconservatively modified variant of SEQ ID NO: 129, a variant of SEQ IDNO: 129 that comprises the canonical structure class of 6, SEQ ID NO:130, a variant of SEQ ID NO: 130, a conservatively modified variant ofSEQ ID NO: 130, a variant of SEQ ID NO: 130 that comprises the canonicalstructure class of 6, SEQ ID NO: 131, a variant of SEQ ID NO: 131, aconservatively modified variant of SEQ ID NO: 131, or a variant of SEQID NO: 131 that comprises the canonical structure class of 3.

The corresponding CDRL2 comprises the amino acid sequence of SEQ ID NO:56, a variant of SEQ ID NO: 56, a conservatively modified variant of SEQID NO: 56, a variant of SEQ ID NO: 56 that comprises the canonicalstructure class of 1, SEQ ID NO: 57, a variant of SEQ ID NO: 57, aconservatively modified variant of SEQ ID NO: 57, a variant of SEQ IDNO: 57 that comprises the canonical structure class of 1, SEQ ID NO: 58,a variant of SEQ ID NO: 58, a conservatively modified variant of SEQ IDNO: 58, a variant of SEQ ID NO: 58 that comprises the canonicalstructure class of 1, SEQ ID NO: 59, a variant of SEQ ID NO: 59, aconservatively modified variant of SEQ ID NO: 59, a variant of SEQ IDNO: 59 that comprises the canonical structure class of 1, SEQ ID NO: 60,a variant of SEQ ID NO: 60, a conservatively modified variant of SEQ IDNO: 60, a variant of SEQ ID NO: 60 that comprises the canonicalstructure class of 1, SEQ ID NO: 61, a variant of SEQ ID NO: 61, aconservatively modified variant of SEQ ID NO: 61, a variant of SEQ IDNO: 61 that comprises the canonical structure class of 1, SEQ ID NO: 62,a variant of SEQ ID NO: 62, a conservatively modified variant of SEQ IDNO: 62, a variant of SEQ ID NO: 62 that comprises the canonicalstructure class of 1, SEQ ID NO: 63, a variant of SEQ ID NO: 63, aconservatively modified variant of SEQ ID NO: 63, a variant of SEQ IDNO: 63 that comprises the canonical structure class of 1, SEQ ID NO: 64,a variant of SEQ ID NO: 64, a conservatively modified variant of SEQ IDNO: 64, or a variant of SEQ ID NO: 64 that comprises the canonicalstructure class of 1, SEQ ID NO: 132, a variant of SEQ ID NO: 132, aconservatively modified variant of SEQ ID NO: 132, a variant of SEQ IDNO: 132 that comprises the canonical structure class of 1, SEQ ID NO:133, a variant of SEQ ID NO: 133, a conservatively modified variant ofSEQ ID NO: 133, a variant of SEQ ID NO: 133 that comprises the canonicalstructure class of 1, SEQ ID NO: 134, a variant of SEQ ID NO: 134, aconservatively modified variant of SEQ ID NO: 134, or a variant of SEQID NO: 134 that comprises the canonical structure class of 1.

The corresponding CDRL3 comprises the amino acid sequence of SEQ ID NO:65, a variant of SEQ ID NO: 65, a conservatively modified variant of SEQID NO: 65, a variant of SEQ ID NO: 65 that comprises the canonicalstructure class of 1, SEQ ID NO: 66, a variant of SEQ ID NO: 66, aconservatively modified variant of SEQ ID NO: 66, a variant of SEQ IDNO: 66 that comprises the canonical structure class of 1, SEQ ID NO: 67,a variant of SEQ ID NO: 67, a conservatively modified variant of SEQ IDNO: 67, a variant of SEQ ID NO: 67 that comprises the canonicalstructure class of 1, SEQ ID NO: 68, a variant of SEQ ID NO: 68, aconservatively modified variant of SEQ ID NO: 68, a variant of SEQ IDNO: 68 that comprises the canonical structure class of 1, SEQ ID NO: 69,a variant of SEQ ID NO: 69, a conservatively modified variant of SEQ IDNO: 69, a variant of SEQ ID NO: 69 that comprises the canonicalstructure class of 1, SEQ ID NO: 70, a variant of SEQ ID NO: 70, aconservatively modified variant of SEQ ID NO: 70, a variant of SEQ IDNO: 70 that comprises the canonical structure class of 1, SEQ ID NO: 71,a variant of SEQ ID NO: 71, a conservatively modified variant of SEQ IDNO: 71, a variant of SEQ ID NO: 71 that comprises the canonicalstructure class of 1, SEQ ID NO: 72, a variant of SEQ ID NO: 72, aconservatively modified variant of SEQ ID NO: 72, a variant of SEQ IDNO: 72 that comprises the canonical structure class of 1, SEQ ID NO: 73,a variant of SEQ ID NO: 73, a conservatively modified variant of SEQ IDNO: 73, a variant of SEQ ID NO: 73 that comprises the canonicalstructure class of 1,

SEQ ID NO: 135, a variant of SEQ ID NO: 135, a conservatively modifiedvariant of SEQ ID NO: 135, a variant of SEQ ID NO: 135 that comprisesthe canonical structure class of 1, SEQ ID NO: 136, a variant of SEQ IDNO: 136, a conservatively modified variant of SEQ ID NO: 136, a variantof SEQ ID NO: 136 that comprises the canonical structure class of 1, SEQID NO: 137, a variant of SEQ ID NO: 137, a conservatively modifiedvariant of SEQ ID NO: 137, a variant of SEQ ID NO: 137 that comprisesthe canonical structure class of 1, SEQ ID NO: 138, a variant of SEQ IDNO: 138, a conservatively modified variant of SEQ ID NO: 138, a variantof SEQ ID NO: 138 that comprises the canonical structure class of 3, SEQID NO: 139, a variant of SEQ ID NO: 139, a conservatively modifiedvariant of SEQ ID NO: 139, or a variant of SEQ ID NO: 139 that comprisesthe canonical structure class of 1.

The corresponding CDRH1 comprises the amino acid sequence of SEQ ID NO:74, a variant of SEQ ID NO: 74, a conservatively modified variant of SEQID NO: 74, a variant of SEQ ID NO: 74 that comprises the canonicalstructure class of 1, SEQ ID NO: 75, a variant of SEQ ID NO: 75, aconservatively modified variant of SEQ ID NO: 75, a variant of SEQ IDNO: 75 that comprises the canonical structure class of 1, SEQ ID NO: 76,a variant of SEQ ID NO: 76, a conservatively modified variant of SEQ IDNO: 76, or a variant of SEQ ID NO: 76 that comprises the canonicalstructure class of 1, SEQ ID NO: 77, a variant of SEQ ID NO: 77, aconservatively modified variant of SEQ ID NO: 77, or a variant of SEQ IDNO: 77 that comprises the canonical structure class of 1, SEQ ID NO: 78,a variant of SEQ ID NO: 78, a conservatively modified variant of SEQ IDNO: 78, a variant of SEQ ID NO: 78 that comprises the canonicalstructure class of 1, SEQ ID NO: 79, a variant of SEQ ID NO: 79, aconservatively modified variant of SEQ ID NO: 79, a variant of SEQ IDNO: 79 that comprises the canonical structure class of 1, SEQ ID NO: 80,a variant of SEQ ID NO: 80, a conservatively modified variant of SEQ IDNO: 80, a variant of SEQ ID NO: 80 that comprises the canonicalstructure class of 1, SEQ ID NO: 81, a variant of SEQ ID NO: 81, aconservatively modified variant of SEQ ID NO: 81, a variant of SEQ IDNO: 81 that comprises the canonical structure class of 1, SEQ ID NO: 82,a variant of SEQ ID NO: 82, a conservatively modified variant of SEQ IDNO: 82, or a variant of SEQ ID NO: 82 that comprises the canonicalstructure class of 1, SEQ ID NO: 140, a variant of SEQ ID NO: 140, aconservatively modified variant of SEQ ID NO: 140, a variant of SEQ IDNO: 140 that comprises the canonical structure class of 1, SEQ ID NO:141, a variant of SEQ ID NO: 141, a conservatively modified variant ofSEQ ID NO: 141, a variant of SEQ ID NO: 141 that comprises the canonicalstructure class of 1, SEQ ID NO: 142, a variant of SEQ ID NO: 142, aconservatively modified variant of SEQ ID NO: 142, a variant of SEQ IDNO: 142 that comprises the canonical structure class of 1, SEQ ID NO:143, a variant of SEQ ID NO: 143, a conservatively modified variant ofSEQ ID NO: 143, or a variant of SEQ ID NO: 143 that comprises thecanonical structure class of 1.

The corresponding CDRH2 comprises the amino acid sequence of SEQ ID NO:83, a variant of SEQ ID NO: 83, a conservatively modified variant of SEQID NO: 83, a variant of SEQ ID NO: 83 that comprises the canonicalstructure class of 3A, SEQ ID NO: 84, a variant of SEQ ID NO: 84, aconservatively modified variant of SEQ ID NO: 84, a variant of SEQ IDNO: 84 that comprises the canonical structure class of 2A, SEQ ID NO:85, a variant of SEQ ID NO: 85, a conservatively modified variant of SEQID NO: 85, or a variant of SEQ ID NO: 85 that comprises the canonicalstructure class of 2B, SEQ ID NO: 86, a variant of SEQ ID NO: 86, aconservatively modified variant of SEQ ID NO: 86, SEQ ID NO: 87, avariant of SEQ ID NO: 87, a conservatively modified variant of SEQ IDNO: 87, a variant of SEQ ID NO: 87 that comprises the canonicalstructure class of 1, SEQ ID NO: 88, a variant of SEQ ID NO: 88, aconservatively modified variant of SEQ ID NO: 88, a variant of SEQ IDNO: 88 that comprises the canonical structure class of 2B, SEQ ID NO:89, a variant of SEQ ID NO: 89, a conservatively modified variant of SEQID NO: 89, a variant of SEQ ID NO: 89 that comprises the canonicalstructure class of 2B, SEQ ID NO: 90, a variant of SEQ ID NO: 90, aconservatively modified variant of SEQ ID NO: 90, a variant of SEQ IDNO: 90 that comprises the canonical structure class of 1, SEQ ID NO: 91,a variant of SEQ ID NO: 91, a conservatively modified variant of SEQ IDNO: 91, a variant of SEQ ID NO: 91 that comprises the canonicalstructure class of 2A, SEQ ID NO: 144, a variant of SEQ ID NO: 144, aconservatively modified variant of SEQ ID NO: 144, a variant of SEQ IDNO: 144 that comprises the canonical structure class of 3A, SEQ ID NO:145, a variant of SEQ ID NO: 145, a conservatively modified variant ofSEQ ID NO: 145, a variant of SEQ ID NO: 145 that comprises the canonicalstructure class of 2A, SEQ ID NO: 146, a variant of SEQ ID NO: 146, aconservatively modified variant of SEQ ID NO: 146, a variant of SEQ IDNO: 146 that comprises the canonical structure class of 3A, SEQ ID NO:147, a variant of SEQ ID NO: 147, a conservatively modified variant ofSEQ ID NO: 147, a variant of SEQ ID NO: 147 that comprises the canonicalstructure class of 3A, SEQ ID NO: 148, a variant of SEQ ID NO: 148, aconservatively modified variant of SEQ ID NO: 148, or a variant of SEQID NO: 148 that comprises the canonical structure class of 3A.

The corresponding CDRH3 comprises the amino acid sequence of SEQ ID NO:92, a variant of SEQ ID NO: 92, a conservatively modified variant of SEQID NO: 92, a variant of SEQ ID NO: 92 that comprises the canonicalstructure class of 12, SEQ ID NO: 93, a variant of SEQ ID NO: 93, aconservatively modified variant of SEQ ID NO: 93, a variant of SEQ IDNO: 93 that comprises the canonical structure class of 7, SEQ ID NO: 94,a variant of SEQ ID NO: 94, a conservatively modified variant of SEQ IDNO: 94, or a variant of SEQ ID NO: 94 that comprises the canonicalstructure class of 15, SEQ ID NO: 95, a variant of SEQ ID NO: 95, aconservatively modified variant of SEQ ID NO: 95, or a variant of SEQ IDNO: 95 that comprises the canonical structure class of 11, SEQ ID NO:96, a variant of SEQ ID NO: 96, a conservatively modified variant of SEQID NO: 96, a variant of SEQ ID NO: 96 that comprises the canonicalstructure class of 15, SEQ ID NO: 97, a variant of SEQ ID NO: 97, aconservatively modified variant of SEQ ID NO: 97, a variant of SEQ IDNO: 97 that comprises the canonical structure class of 6, SEQ ID NO: 98,a variant of SEQ ID NO: 98, a conservatively modified variant of SEQ IDNO: 98, a variant of SEQ ID NO: 98 that comprises the canonicalstructure class of 4, SEQ ID NO: 99, a variant of SEQ ID NO: 99, aconservatively modified variant of SEQ ID NO: 99, a variant of SEQ IDNO: 99 that comprises the canonical structure class of 13, SEQ ID NO:100, a variant of SEQ ID NO: 100, a conservatively modified variant ofSEQ ID NO: 100, or a variant of SEQ ID NO: 100 that comprises thecanonical structure class of 6, SEQ ID NO: 149, a variant of SEQ ID NO:149, a conservatively modified variant of SEQ ID NO: 149, a variant ofSEQ ID NO: 149 that comprises the canonical structure class of 15, SEQID NO: 150, a variant of SEQ ID NO: 150, a conservatively modifiedvariant of SEQ ID NO: 150, a variant of SEQ ID NO: 150 that comprisesthe canonical structure class of 10, SEQ ID NO: 151, a variant of SEQ IDNO: 151, a conservatively modified variant of SEQ ID NO: 151, a variantof SEQ ID NO: 151 that comprises the canonical structure class of 15,SEQ ID NO: 152, a variant of SEQ ID NO: 152, a conservatively modifiedvariant of SEQ ID NO: 152, a variant of SEQ ID NO: 152 that comprisesthe canonical structure class of 9, SEQ ID NO: 153, a variant of SEQ IDNO: 153, a conservatively modified variant of SEQ ID NO: 153, or avariant of SEQ ID NO: 153 that comprises the canonical structure classof 13.

In particular embodiments the mammalian antibodies (including chimericmammalian antibodies) and/or antigen binding fragments thereof of thepresent invention bind the canine interleukin-4 receptor alpha(IL-4R_(a)) and/or block the binding of canine IL-4R_(a), to canine IL-4and/or canine IL-13. In related embodiments the mammalian antibodiesand/or antigen binding fragments thereof of the present invention blockthe binding of canine IL-4 and/or canine IL-13 to the IL-4 Type Ireceptor and/or the IL-4 Type II receptor. In particular embodiments themammalian antibodies (whether isolated or not) are caninized antibodies.

Accordingly, in certain embodiments of mammalian antibodies (includingcaninized antibodies) the CDRL1 comprises the amino acid sequence of SEQID NO: 47, a variant of SEQ ID NO: 47, a conservatively modified variantof SEQ ID NO: 47, or a variant of SEQ ID NO: 47 that comprises thecanonical structure class of 1; the CDRL2 comprises the amino acidsequence of SEQ ID NO: 56, a variant of SEQ ID NO: 56, a conservativelymodified variant of SEQ ID NO: 56, or a variant of SEQ ID NO: 56 thatcomprises the canonical structure class of 1; the CDRL3 comprises theamino acid sequence of SEQ ID NO: 65, a variant of SEQ ID NO: 65, aconservatively modified variant of SEQ ID NO: 65, or a variant of SEQ IDNO: 65 that comprises the canonical structure class of 1, the CDRH1comprises the amino acid sequence of SEQ ID NO: 74, a variant of SEQ IDNO: 74, a conservatively modified variant of SEQ ID NO: 74, or a variantof SEQ ID NO: 74 that comprises the canonical structure class of 1; theCDRH2 comprises the amino acid sequence of SEQ ID NO: 83, a variant ofSEQ ID NO: 83, a conservatively modified variant of SEQ ID NO: 83, and avariant of SEQ ID NO: 83 that comprises the canonical structure class of3A, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 92, avariant of SEQ ID NO: 92, a conservatively modified variant of SEQ IDNO: 92, or a variant of SEQ ID NO: 92 that comprises the canonicalstructure class of 12.

In yet other embodiments of mammalian antibodies (including caninizedantibodies) the CDRL1 comprises the amino acid sequence of SEQ ID NO:48, a variant of SEQ ID NO: 48, a conservatively modified variant of SEQID NO: 48, or a variant of SEQ ID NO: 48 that comprises the canonicalstructure class of 2A; the CDRL2 comprises the amino acid sequence ofSEQ ID NO: 57, a variant of SEQ ID NO: 57, a conservatively modifiedvariant of SEQ ID NO: 57, or a variant of SEQ ID NO: 57 that comprisesthe canonical structure class of 1; the CDRL3 comprises the amino acidsequence of SEQ ID NO: 66, a variant of SEQ ID NO: 66, a conservativelymodified variant of SEQ ID NO: 66, or a variant of SEQ ID NO: 66 thatcomprises the canonical structure class of 1, the CDRH1 comprises theamino acid sequence of SEQ ID NO: 75, a variant of SEQ ID NO: 75, aconservatively modified variant of SEQ ID NO: 75, or a variant of SEQ IDNO: 75 that comprises the canonical structure class of 1; the CDRH2comprises the amino acid sequence of SEQ ID NO: 84, a variant of SEQ IDNO: 84, a conservatively modified variant of SEQ ID NO: 84, and avariant of SEQ ID NO: 84 that comprises the canonical structure class of2A, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 93, avariant of SEQ ID NO: 93, a conservatively modified variant of SEQ IDNO: 93, or a variant of SEQ ID NO: 93 that comprises the canonicalstructure class of 7.

In still other embodiments of mammalian antibodies (including caninizedantibodies) the CDRL1 comprises the amino acid sequence of SEQ ID NO:49, a variant of SEQ ID NO: 49, a conservatively modified variant of SEQID NO: 49, or a variant of SEQ ID NO: 49 that comprises the canonicalstructure class of 4; the CDRL2 comprises the amino acid sequence of SEQID NO: 58, a variant of SEQ ID NO: 58, a conservatively modified variantof SEQ ID NO: 58, or a variant of SEQ ID NO: 58 that comprises thecanonical structure class of 4; the CDRL3 comprises the amino acidsequence of SEQ ID NO: 67, a variant of SEQ ID NO: 67, a conservativelymodified variant of SEQ ID NO: 67, or a variant of SEQ ID NO: 67 thatcomprises the canonical structure class of 1, the CDRH1 comprises theamino acid sequence of SEQ ID NO: 76, a variant of SEQ ID NO: 76, aconservatively modified variant of SEQ ID NO: 76, or a variant of SEQ IDNO: 76 that comprises the canonical structure class of 1; the CDRH2comprises the amino acid sequence of SEQ ID NO: 85, a variant of SEQ IDNO: 85, a conservatively modified variant of SEQ ID NO: 85, and avariant of SEQ ID NO: 85 that comprises the canonical structure class of2B, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 94, avariant of SEQ ID NO: 94, a conservatively modified variant of SEQ IDNO: 94, or a variant of SEQ ID NO: 94 that comprises the canonicalstructure class of 15.

In yet other embodiments of mammalian antibodies (including caninizedantibodies) the CDRL1 comprises the amino acid sequence of SEQ ID NO:51, a variant of SEQ ID NO: 51, a conservatively modified variant of SEQID NO: 51, or a variant of SEQ ID NO: 51 that comprises the canonicalstructure class of 3; the CDRL2 comprises the amino acid sequence of SEQID NO: 60, a variant of SEQ ID NO: 60, a conservatively modified variantof SEQ ID NO: 60, or a variant of SEQ ID NO: 60 that comprises thecanonical structure class of 1; the CDRL3 comprises the amino acidsequence of SEQ ID NO: 69, a variant of SEQ ID NO: 69, a conservativelymodified variant of SEQ ID NO: 69, or a variant of SEQ ID NO: 69 thatcomprises the canonical structure class of 1, the CDRH1 comprises theamino acid sequence of SEQ ID NO: 78, a variant of SEQ ID NO: 78, aconservatively modified variant of SEQ ID NO: 78, or a variant of SEQ IDNO: 78 that comprises the canonical structure class of 1; the CDRH2comprises the amino acid sequence of SEQ ID NO: 87, a variant of SEQ IDNO: 87, a conservatively modified variant of SEQ ID NO: 87, and avariant of SEQ ID NO: 87 that comprises the canonical structure class of1, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 96, avariant of SEQ ID NO: 96, a conservatively modified variant of SEQ IDNO: 96, or a variant of SEQ ID NO: 96 that comprises the canonicalstructure class of 15.

In still other embodiments of mammalian antibodies (including caninizedantibodies) the CDRL1 comprises the amino acid sequence of SEQ ID NO:52, a variant of SEQ ID NO: 52, a conservatively modified variant of SEQID NO: 52, or a variant of SEQ ID NO: 52 that comprises the canonicalstructure class of 2A; the CDRL2 comprises the amino acid sequence ofSEQ ID NO: 61, a variant of SEQ ID NO: 61, a conservatively modifiedvariant of SEQ ID NO: 61, or a variant of SEQ ID NO: 61 that comprisesthe canonical structure class of 1; the CDRL3 comprises the amino acidsequence of SEQ ID NO: 70, a variant of SEQ ID NO: 70, a conservativelymodified variant of SEQ ID NO: 70, or a variant of SEQ ID NO: 70 thatcomprises the canonical structure class of 1, the CDRH1 comprises theamino acid sequence of SEQ ID NO: 79, a variant of SEQ ID NO: 79, aconservatively modified variant of SEQ ID NO: 79, or a variant of SEQ IDNO: 79 that comprises the canonical structure class of 1; the CDRH2comprises the amino acid sequence of SEQ ID NO: 88, a variant of SEQ IDNO: 88, a conservatively modified variant of SEQ ID NO: 88, and avariant of SEQ ID NO: 88 that comprises the canonical structure class of2B, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 97, avariant of SEQ ID NO: 97, a conservatively modified variant of SEQ IDNO: 97, or a variant of SEQ ID NO: 97 that comprises the canonicalstructure class of 6.

In yet other embodiments of mammalian antibodies (including caninizedantibodies) the CDRL1 comprises the amino acid sequence of SEQ ID NO:53, a variant of SEQ ID NO: 53, a conservatively modified variant of SEQID NO: 53, or a variant of SEQ ID NO: 53 that comprises the canonicalstructure class of 6; the CDRL2 comprises the amino acid sequence of SEQID NO: 62, a variant of SEQ ID NO: 62, a conservatively modified variantof SEQ ID NO: 62, or a variant of SEQ ID NO: 62 that comprises thecanonical structure class of 1; the CDRL3 comprises the amino acidsequence of SEQ ID NO: 71, a variant of SEQ ID NO: 71, a conservativelymodified variant of SEQ ID NO: 71, or a variant of SEQ ID NO: 71 thatcomprises the canonical structure class of 1, the CDRH1 comprises theamino acid sequence of SEQ ID NO: 80, a variant of SEQ ID NO: 80, aconservatively modified variant of SEQ ID NO: 80, or a variant of SEQ IDNO: 80 that comprises the canonical structure class of 1; the CDRH2comprises the amino acid sequence of SEQ ID NO: 89, a variant of SEQ IDNO: 89, a conservatively modified variant of SEQ ID NO: 89, and avariant of SEQ ID NO: 89 that comprises the canonical structure class of2B, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 98, avariant of SEQ ID NO: 98, a conservatively modified variant of SEQ IDNO: 98, or a variant of SEQ ID NO: 98 that comprises the canonicalstructure class of 4.

In still other embodiments of mammalian antibodies (including caninizedantibodies) the CDRL1 comprises the amino acid sequence of SEQ ID NO:54, a variant of SEQ ID NO: 54, a conservatively modified variant of SEQID NO: 54, or a variant of SEQ ID NO: 54 that comprises the canonicalstructure class of 1; the CDRL2 comprises the amino acid sequence of SEQID NO: 63, a variant of SEQ ID NO: 63, a conservatively modified variantof SEQ ID NO: 63, or a variant of SEQ ID NO: 63 that comprises thecanonical structure class of 1; the CDRL3 comprises the amino acidsequence of SEQ ID NO: 72, a variant of SEQ ID NO: 72, a conservativelymodified variant of SEQ ID NO: 72, or a variant of SEQ ID NO: 72 thatcomprises the canonical structure class of 1, the CDRH1 comprises theamino acid sequence of SEQ ID NO: 81, a variant of SEQ ID NO: 81, aconservatively modified variant of SEQ ID NO: 81, or a variant of SEQ IDNO: 81 that comprises the canonical structure class of 1; the CDRH2comprises the amino acid sequence of SEQ ID NO: 90, a variant of SEQ IDNO: 90, a conservatively modified variant of SEQ ID NO: 90, and avariant of SEQ ID NO: 90 that comprises the canonical structure class of1, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 99, avariant of SEQ ID NO: 99, a conservatively modified variant of SEQ IDNO: 99, or a variant of SEQ ID NO: 99 that comprises the canonicalstructure class of 13. In particular embodiments of this type, when theantibody (or antigen binding fragment thereof) binds canineinterleukin-4 receptor α (IL-4R_(a)) the antibody binds to at least oneamino acid residue, preferably two to five amino acid residues, and/ormore preferably three to eight amino acid residues or more within theamino acid sequence of SEQ ID NO: 157, or SEQ ID NO: 158, or within bothSEQ ID NO: 157 and SEQ ID NO: 158.

In yet other embodiments of mammalian antibodies (including caninizedantibodies) the CDRL1 comprises the amino acid sequence of SEQ ID NO:55, a variant of SEQ ID NO: 55, a conservatively modified variant of SEQID NO: 55, or a variant of SEQ ID NO: 55 that comprises the canonicalstructure class of 2A; the CDRL2 comprises the amino acid sequence ofSEQ ID NO: 64, a variant of SEQ ID NO: 64, a conservatively modifiedvariant of SEQ ID NO: 64, or a variant of SEQ ID NO: 64 that comprisesthe canonical structure class of 1; the CDRL3 comprises the amino acidsequence of SEQ ID NO: 73, a variant of SEQ ID NO: 73, a conservativelymodified variant of SEQ ID NO: 73, or a variant of SEQ ID NO: 73 thatcomprises the canonical structure class of 1, the CDRH1 comprises theamino acid sequence of SEQ ID NO: 82, a variant of SEQ ID NO: 82, aconservatively modified variant of SEQ ID NO: 82, or a variant of SEQ IDNO: 82 that comprises the canonical structure class of 1; the CDRH2comprises the amino acid sequence of SEQ ID NO: 91, a variant of SEQ IDNO: 91, a conservatively modified variant of SEQ ID NO: 91, and avariant of SEQ ID NO: 91 that comprises the canonical structure class of2A, the CDRH3 comprises the amino acid sequence of SEQ ID NO: 100, avariant of SEQ ID NO: 100, a conservatively modified variant of SEQ IDNO: 100, or a variant of SEQ ID NO: 100 that comprises the canonicalstructure class of 6.

In still other embodiments of mammalian antibodies (including caninizedantibodies) the CDRL1 comprises the amino acid sequence of SEQ ID NO:129, a variant of SEQ ID NO: 129, a conservatively modified variant ofSEQ ID NO: 129, or a variant of SEQ ID NO: 129 that comprises thecanonical structure class of 6; the CDRL2 comprises the amino acidsequence of SEQ ID NO: 132, a variant of SEQ ID NO: 132, aconservatively modified variant of SEQ ID NO: 132, or a variant of SEQID NO: 132 that comprises the canonical structure class of 1; the CDRL3comprises the amino acid sequence of SEQ ID NO: 135, a variant of SEQ IDNO: 135, a conservatively modified variant of SEQ ID NO: 135, or avariant of SEQ ID NO: 135 that comprises the canonical structure classof 1, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 140, avariant of SEQ ID NO: 140, a conservatively modified variant of SEQ IDNO: 140, or a variant of SEQ ID NO: 140 that comprises the canonicalstructure class of 1; the CDRH2 comprises the amino acid sequence of SEQID NO: 144, a variant of SEQ ID NO: 144, a conservatively modifiedvariant of SEQ ID NO: 144, and a variant of SEQ ID NO: 144 thatcomprises the canonical structure class of 3A, the CDRH3 comprises theamino acid sequence of SEQ ID NO: 149, a variant of SEQ ID NO: 149, aconservatively modified variant of SEQ ID NO: 149, or a variant of SEQID NO: 149 that comprises the canonical structure class of 15. Inparticular embodiments of this type, when the antibody (or antigenbinding fragment thereof) binds canine interleukin-4 receptor α(IL-4R_(a)) the antibody binds to at least one amino acid residue,preferably two to five amino acid residues, and/or more preferably threeto eight amino acid residues or more within the amino acid sequence ofSEQ ID NO: 127, or SEQ ID NO: 128, or within both SEQ ID NO: 127 and SEQID NO: 128.

In yet other embodiments of mammalian antibodies (including caninizedantibodies) the CDRL1 comprises the amino acid sequence of SEQ ID NO:130, a variant of SEQ ID NO: 130, a conservatively modified variant ofSEQ ID NO: 130, or a variant of SEQ ID NO: 130 that comprises thecanonical structure class of 6; the CDRL2 comprises the amino acidsequence of SEQ ID NO: 133, a variant of SEQ ID NO: 133, aconservatively modified variant of SEQ ID NO: 133, or a variant of SEQID NO: 133 that comprises the canonical structure class of 1; the CDRL3comprises the amino acid sequence of SEQ ID NO: 136, a variant of SEQ IDNO: 136, a conservatively modified variant of SEQ ID NO: 136, or avariant of SEQ ID NO: 136 that comprises the canonical structure classof 1, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 141, avariant of SEQ ID NO: 141, a conservatively modified variant of SEQ IDNO: 141, or a variant of SEQ ID NO: 141 that comprises the canonicalstructure class of 1; the CDRH2 comprises the amino acid sequence of SEQID NO: 145, a variant of SEQ ID NO: 145, a conservatively modifiedvariant of SEQ ID NO: 145, and a variant of SEQ ID NO: 145 thatcomprises the canonical structure class of 2A, the CDRH3 comprises theamino acid sequence of SEQ ID NO: 150, a variant of SEQ ID NO: 150, aconservatively modified variant of SEQ ID NO: 150, or a variant of SEQID NO: 150 that comprises the canonical structure class of 10. Inparticular embodiments of this type, when the antibody (or antigenbinding fragment thereof) binds canine interleukin-4 receptor α(IL-4R_(a)) the antibody binds to at least one amino acid residue,preferably two to five amino acid residues, and/or more preferably threeto eight amino acid residues or more within the amino acid sequence ofSEQ ID NO: 158, or SEQ ID NO: 162, or within both SEQ ID NO: 158 and SEQID NO: 162.

In still other embodiments of mammalian antibodies (including caninizedantibodies) the CDRL1 comprises the amino acid sequence of SEQ ID NO:129, a variant of SEQ ID NO: 129, a conservatively modified variant ofSEQ ID NO: 129, or a variant of SEQ ID NO: 129 that comprises thecanonical structure class of 6; the CDRL2 comprises the amino acidsequence of SEQ ID NO: 134, a variant of SEQ ID NO: 134, aconservatively modified variant of SEQ ID NO: 134, or a variant of SEQID NO: 134 that comprises the canonical structure class of 1; the CDRL3comprises the amino acid sequence of SEQ ID NO: 137, a variant of SEQ IDNO: 137, a conservatively modified variant of SEQ ID NO: 137, or avariant of SEQ ID NO: 137 that comprises the canonical structure classof 1, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 140, avariant of SEQ ID NO: 140, a conservatively modified variant of SEQ IDNO: 140, or a variant of SEQ ID NO: 140 that comprises the canonicalstructure class of 1; the CDRH2 comprises the amino acid sequence of SEQID NO: 146, a variant of SEQ ID NO: 146, a conservatively modifiedvariant of SEQ ID NO: 146, and a variant of SEQ ID NO: 146 thatcomprises the canonical structure class of 3A, the CDRH3 comprises theamino acid sequence of SEQ ID NO: 151, a variant of SEQ ID NO: 151, aconservatively modified variant of SEQ ID NO: 151, or a variant of SEQID NO: 151 that comprises the canonical structure class of 15. Inparticular embodiments of this type, when the antibody (or antigenbinding fragment thereof) binds canine interleukin-4 receptor α(IL-4R_(a)) the antibody binds to at least one amino acid residue,preferably two to five amino acid residues, and/or more preferably threeto eight amino acid residues or more within the amino acid sequence ofSEQ ID NO: 125 or SEQ ID NO: 126, or within both SEQ ID NO: 125 and SEQID NO: 126.

In yet other embodiments of mammalian antibodies (including caninizedantibodies) the CDRL1 comprises the amino acid sequence of SEQ ID NO:131, a variant of SEQ ID NO: 131, a conservatively modified variant ofSEQ ID NO: 131, or a variant of SEQ ID NO: 131 that comprises thecanonical structure class of 3; the CDRL2 comprises the amino acidsequence of SEQ ID NO: 60, a variant of SEQ ID NO: 60, a conservativelymodified variant of SEQ ID NO: 60, or a variant of SEQ ID NO: 60 thatcomprises the canonical structure class of 1; the CDRL3 comprises theamino acid sequence of SEQ ID NO: 138, a variant of SEQ ID NO: 138, aconservatively modified variant of SEQ ID NO: 1385, or a variant of SEQID NO: 138 that comprises the canonical structure class of 3, the CDRH1comprises the amino acid sequence of SEQ ID NO: 142, a variant of SEQ IDNO: 142, a conservatively modified variant of SEQ ID NO: 142, or avariant of SEQ ID NO: 142 that comprises the canonical structure classof 1; the CDRH2 comprises the amino acid sequence of SEQ ID NO: 147, avariant of SEQ ID NO: 147, a conservatively modified variant of SEQ IDNO: 147, and a variant of SEQ ID NO: 147 that comprises the canonicalstructure class of 3A, the CDRH3 comprises the amino acid sequence ofSEQ ID NO: 152, a variant of SEQ ID NO: 152, a conservatively modifiedvariant of SEQ ID NO: 152, or a variant of SEQ ID NO: 152 that comprisesthe canonical structure class of 9. In particular embodiments of thistype, when the antibody (or antigen binding fragment thereof) bindscanine interleukin-4 receptor α (IL-4R_(a)) the antibody binds to atleast one amino acid residue, preferably two to five amino acidresidues, and/or more preferably three to eight amino acid residues ormore within the amino acid sequence of SEQ ID NO: 154, or SEQ ID NO:155, or SEQ ID NO: 156, or any combination thereof.

In still other embodiments of mammalian antibodies (including caninizedantibodies) the CDRL1 comprises the amino acid sequence of SEQ ID NO:129, a variant of SEQ ID NO: 129, a conservatively modified variant ofSEQ ID NO: 129, or a variant of SEQ ID NO: 129 that comprises thecanonical structure class of 6; the CDRL2 comprises the amino acidsequence of SEQ ID NO: 132, a variant of SEQ ID NO: 132, aconservatively modified variant of SEQ ID NO: 132, or a variant of SEQID NO: 132 that comprises the canonical structure class of 1; the CDRL3comprises the amino acid sequence of SEQ ID NO: 139, a variant of SEQ IDNO: 139, a conservatively modified variant of SEQ ID NO: 139, or avariant of SEQ ID NO: 139 that comprises the canonical structure classof 1, the CDRH1 comprises the amino acid sequence of SEQ ID NO: 143, avariant of SEQ ID NO: 143, a conservatively modified variant of SEQ IDNO: 143, or a variant of SEQ ID NO: 143 that comprises the canonicalstructure class of 1; the CDRH2 comprises the amino acid sequence of SEQID NO: 148, a variant of SEQ ID NO: 148, a conservatively modifiedvariant of SEQ ID NO: 148, and a variant of SEQ ID NO: 148 thatcomprises the canonical structure class of 3A, the CDRH3 comprises theamino acid sequence of SEQ ID NO: 153, a variant of SEQ ID NO: 153, aconservatively modified variant of SEQ ID NO: 153, or a variant of SEQID NO: 153 that comprises the canonical structure class of 13. Inparticular embodiments of this type, when the antibody (or antigenbinding fragment thereof) binds canine interleukin-4 receptor α(IL-4R_(a)) the antibody binds to at least one amino acid residue,preferably two to five amino acid residues, and/or more preferably threeto eight amino acid residues or more within the amino acid sequence ofSEQ ID NO: 159, or SEQ ID NO: 160, or SEQ ID NO: 161, or any combinationthereof.

The present invention includes antibodies and antigen binding fragmentsthereof that bind canine interleukin-4 receptor alpha (IL-4R_(a)) withspecificity. In particular embodiments of this type, the antibodies andantigen binding fragments thereof bind canine IL-4R_(a) and block thebinding of canine IL-4R_(a) to canine IL-4 and/or IL-13. As indicatedabove, the isolated mammalian antibodies or antigen binding fragmentsthereof can be caninized antibodies or caninized antigen bindingfragments thereof. In other embodiments, the isolated mammalianantibodies or antigen binding fragments thereof can be murine antibodiesor murine antigen binding fragments thereof.

The caninized antibodies or caninized antigen binding fragments thereofof the present invention can comprise a hinge region. In a particularembodiment of this type, the hinge region comprises the amino acidsequence of SEQ ID NO: 101. In another embodiment the hinge regioncomprises the amino acid sequence of SEQ ID NO: 102. In still anotherembodiment the hinge region comprises the amino acid sequence of SEQ IDNO: 103. In yet another embodiment the hinge region comprises the aminoacid sequence of SEQ ID NO: 104.

In certain embodiments the caninized antibody or antigen bindingfragment thereof, comprises a heavy chain that comprises the amino acidsequence of SEQ ID NO: 164. In particular embodiments of this type, theheavy chain is encoded by the nucleotide sequence of SEQ ID NO: 163. Inother embodiments the caninized antibody or antigen binding fragmentthereof, comprises a heavy chain that comprises the amino acid sequenceof SEQ ID NO: 166. In particular embodiments of this type, the heavychain is encoded by the nucleotide sequence of SEQ ID NO: 165. In stillother embodiments, the caninized antibody or antigen binding fragmentthereof, comprises a heavy chain that comprises the amino acid sequenceof SEQ ID NO: 168. In particular embodiments of this type, the heavychain is encoded by the nucleotide sequence of SEQ ID NO: 167. Inspecific embodiments of such types, when the caninized antibody (orantigen binding fragment thereof) binds canine interleukin-4 receptor α(IL-4R_(a)) the antibody binds to at least one amino acid residue,preferably two to five amino acid residues, and/or more preferably threeto eight amino acid residues or more within the amino acid sequence ofSEQ ID NO: 154, or SEQ ID NO: 155, or SEQ ID NO: 156, or any combinationthereof.

In related embodiments the caninized antibody or antigen bindingfragment thereof, comprises a light chain that comprises the amino acidsequence of SEQ ID NO: 170. In particular embodiments of this type, thelight chain is encoded by the nucleotide sequence of SEQ ID NO: 169. Inother embodiments the caninized antibody or antigen binding fragmentthereof, comprises a light chain comprising the amino acid sequence ofSEQ ID NO: 172. In particular embodiments of this type, the light chainis encoded by the nucleotide sequence of SEQ ID NO: 171. In yet otherembodiments the caninized antibody or antigen binding fragment thereof,comprises a light chain comprising the amino acid sequence of SEQ ID NO:174. In particular embodiments of this type, the light chain is encodedby the nucleotide sequence of SEQ ID NO: 173. In particular embodimentsof such types, when the caninized antibody (or antigen binding fragmentthereof) binds canine interleukin-4 receptor α (IL-4R_(a)) the antibodybinds to at least one amino acid residue, preferably two to five aminoacid residues, and/or more preferably three to eight amino acid residuesor more within the amino acid sequence of SEQ ID NO: 154, or SEQ ID NO:155, or SEQ ID NO: 156, or any combination thereof.

The present invention further provides antibodies comprising acombination of such heavy chains and light chains. In particularembodiments the heavy chain comprises the amino acid sequence of SEQ IDNO: 164 and the light chain comprises the amino acid sequence of SEQ IDNO: 170. In more particular embodiments of this type, the heavy chain isencoded by the nucleotide sequence of SEQ ID NO: 163 and the light chainis encoded by the nucleotide sequence of SEQ ID NO: 169. In otherembodiments the heavy chain comprises the amino acid sequence of SEQ IDNO: 166 and the light chain comprises the amino acid sequence of SEQ IDNO: 172. In more particular embodiments of this type, the heavy chain isencoded by the nucleotide sequence of SEQ ID NO: 165 and the light chainis encoded by the nucleotide sequence of SEQ ID NO: 171. In still otherembodiments the heavy chain comprises the amino acid sequence of SEQ IDNO: 168 and the light chain comprises the amino acid sequence of SEQ IDNO: 174. In more particular embodiments of this type, the heavy chain isencoded by the nucleotide sequence of SEQ ID NO: 167 and the light chainis encoded by the nucleotide sequence of SEQ ID NO: 173.

In related embodiments the heavy chain comprises the amino acid sequenceof SEQ ID NO: 164 and the light chain comprises the amino acid sequenceof SEQ ID NO: 172. In other embodiments the heavy chain comprises theamino acid sequence of SEQ ID NO: 164 and the light chain comprises theamino acid sequence of SEQ ID NO: 174. In still other embodiments theheavy chain comprises the amino acid sequence of SEQ ID NO: 166 and thelight chain comprises the amino acid sequence of SEQ ID NO: 170. In yetother embodiments the heavy chain comprises the amino acid sequence ofSEQ ID NO: 166 and the light chain comprises the amino acid sequence ofSEQ ID NO: 174. In still other embodiments the heavy chain comprises theamino acid sequence of SEQ ID NO: 168 and the light chain comprises theamino acid sequence of SEQ ID NO: 170. In other embodiments the heavychain comprises the amino acid sequence of SEQ ID NO: 168 and the lightchain comprises the amino acid sequence of SEQ ID NO: 172.

In particular embodiments of such types, when the caninized antibody (orantigen binding fragment thereof) binds canine interleukin-4 receptor α(IL-4R_(α)) the antibody binds to at least one amino acid residue,preferably two to five amino acid residues, and/or more preferably threeto eight or more amino acid residues within the amino acid sequence ofSEQ ID NO: 154, or SEQ ID NO: 155, or SEQ ID NO: 156, or any combinationthereof.

Accordingly, the present invention further provides isolated mammalianantibodies or antigen binding fragments thereof (including caninizedantibodies or antigen binding fragments thereof) that bind canineinterleukin-4 receptor α (IL-4R_(α)) with specificity, and when bound tocanine IL-4R_(a) the antibody binds to at least one amino acid residue,preferably two to five amino acid residues, and/or more preferably threeto eight amino acid residues or more within the amino acid sequence ofSEQ ID NO: 125, or SEQ ID NO: 126, or SEQ ID NO: 127, or SEQ ID NO: 128,or SEQ ID NO: 154, or SEQ ID NO: 155, or SEQ ID NO: 156, or SEQ ID NO:157, or SEQ ID NO: 158, or SEQ ID NO: 159, or SEQ ID NO: 160, or SEQ IDNO: 161, or SEQ ID NO: 162, or any combination thereof. In particularembodiments, the antibody or antigen binding fragment thereof bindscanine IL-4R_(a) and blocks the binding of canine IL-4R_(a) to canineinterleukin-4.

The present invention further provides mammalian antibodies or antigenbinding fragments thereof that bind to canine IL-4R_(a) with adissociation constant (Kd) that is lower (e.g., 1×10⁻¹³ M, or lower)than 1×10⁻¹²M. In particular embodiments the mammalian antibodies orantigen binding fragments thereof bind to canine IL-4R_(a) with adissociation constant of 1×10⁻⁵ M to 1×10⁻¹²M. In more particularembodiments the mammalian antibodies or antigen binding fragmentsthereof bind to canine IL-4R_(a) with a dissociation constant of 1×10⁻⁷M to 1×10⁻¹¹ M. In still more particular embodiments the mammalianantibodies or antigen binding fragments thereof bind to canine IL-4R_(a)with a dissociation constant of 1×10⁻⁸M to 1×10⁻¹¹ M. In yet moreparticular embodiments the mammalian antibodies or antigen bindingfragments thereof bind to canine IL-4R_(a) with a dissociation constantof 1×10⁻⁸M to 1×10⁻¹⁰ M.

The present invention also provides mammalian antibodies or antigenbinding fragments thereof that bind to canine IL-4R_(a) with an on rate(k_(on)) that is greater than 1×10⁷M⁻¹s⁻¹. In particular embodiments themammalian antibodies or antigen binding fragments thereof bind to canineIL-4R_(a) with an on rate of 1×10² M⁻¹s⁻¹ to 1×10⁷M⁻¹s⁻¹. In moreparticular embodiments the mammalian antibodies or antigen bindingfragments thereof bind to canine IL-4R_(α) with an on rate of 1×10³M⁻¹s⁻¹ to 1×10⁶M⁻¹s⁻¹. In still more particular embodiments themammalian antibodies or antigen binding fragments thereof bind to canineIL-4R_(α) with an on rate of 1×10³ M⁻¹s⁻¹ to 1×10⁵M⁻¹s⁻¹. In yet moreparticular embodiments the mammalian antibodies or antigen bindingfragments thereof bind to canine IL-4R_(a) on rate of 1×10⁴ M⁻¹s⁻¹ to1×10⁵M⁻¹s⁻¹.

The present invention further provides mammalian antibodies or antigenbinding fragments thereof that bind to canine IL-4R_(a) with an off rate(k_(off)) slower than 1×10⁻⁷ s⁻¹. In particular embodiments themammalian antibodies or antigen binding fragments thereof bind to canineIL-4R_(a) with an off rate of 1×10⁻³ s⁻¹ to 1×10⁻⁸ s⁻¹. In moreparticular embodiments the mammalian antibodies or antigen bindingfragments thereof bind to canine IL-4R_(a) with an off rate of 1×10⁻⁴s⁻¹ to 1×10⁻⁷ s⁻¹. In still more particular embodiments the mammalianantibodies or antigen binding fragments thereof bind to canine IL-4R_(a)with an off rate of 1×10⁻⁵ s⁻¹ to 1×10⁻⁷ s⁻¹.

In particular embodiments, a mammalian antibody of the present invention(including chimeric antibodies) blocks the binding of canine IL-4 withIL-4R_(a). In more particular embodiments the antibody blocks thebinding of canine IL-4 to IL-4R_(a) with a minimum EC50 of 1×10⁻⁸M to1×10⁻⁹M or an even lower concentration. In still more particularembodiments the EC50 is 5×10⁻⁹M to 5×10⁻¹³M. In still more particularembodiments the EC50 is between 5×10⁻⁹M and 5×10⁻¹¹M.

In related embodiments, the mammalian antibodies or antigen bindingfragments thereof negatively attenuate, e.g., inhibit, the cellsignaling pathway(s) mediated by IL-4 and/or IL-13 binding to type Iand/or type II IL-4 receptors. In particular embodiments, the mammalianantibodies or antigen binding fragments thereof ameliorate a pruriticinflammatory skin disease, e.g., atopic dermatitis, in an animalsubject. In more specific embodiments the animal subject is a canine. Ina related embodiment, the animal subject is a feline.

Accordingly, any of the antibodies of the present invention can exhibitone, two, three, four, or all these properties, i.e., the aforesaiddissociation constants with canine IL-4R_(a), the aforesaid on rates forbinding with canine IL-4R_(a), the aforesaid off rates for dissociatingfrom the antibody-canine IL-4R_(a) binding complex, inhibiting the cellsignaling pathway(s) mediated by IL-4 and/or IL-13 binding to type Iand/or type II IL-4 receptors, or ameliorating a pruritic inflammatoryskin disease, e.g., atopic dermatitis, in an animal subject.

As indicated above, the antibodies (and antigen binding fragmentsthereof) of the present invention, including the aforesaid antibodies(and antigen binding fragments thereof), can be monoclonal antibodies(and antigen binding fragments thereof), mammalian antibodies (andantigen binding fragments thereof), e.g., murine (mouse) antibodies (andantigen binding fragments thereof), caninized antibodies (and antigenbinding fragments thereof) including caninized murine antibodies (andantigen binding fragments thereof), and in certain embodiments theantibodies (and antigen binding fragments thereof) are isolated.

The present invention further provides nucleic acids (including isolatednucleic acids) that encode any one of the light chains of the caninizedantibody of the present invention. Similarly, the present inventionprovides isolated nucleic acids that encode any one of the heavy chainsof the caninized antibody of the present invention.

The present invention further provides expression vectors that compriseone or more of the nucleic acids (including isolated nucleic acids) ofthe present invention. The present invention further provides host cellsthat comprise one or more expression vectors of the present invention.

In particular embodiments, the antibody is a recombinant antibody or anantigen binding fragment thereof. In related embodiments, the variableheavy chain domain and variable light chain domain are connected by aflexible linker to form a single-chain antibody.

In particular embodiments, the antibody or antigen binding fragment is aFab fragment. In other embodiments, the antibody or antigen bindingfragment is a Fab′ fragment. In other embodiments, the antibody orantigen binding fragment is a (Fab′)₂ fragment. In still otherembodiments, the antibody or antigen binding fragment is a diabody. Inparticular embodiments, the antibody or antigen binding fragment is adomain antibody. In particular embodiments, the antibody or antigenbinding fragment is a single domain antibody.

In particular embodiments, a caninized murine anti-canine IL-4R_(a)antibody or antigen binding fragment negatively attenuates the cellsignaling pathway(s) mediated by IL-4 and/or IL-13 binding to type Iand/or type II IL-4 receptors in an animal subject (e.g., canine) beingtreated. In more particular embodiments, administration of a caninizedmurine anti-canine IL-4R_(a) antibody or antigen binding fragment of thepresent invention serves to ameliorate one or more symptom of atopicdermatitis in the animal subject (e.g., canine) being treated.

The present invention further provides isolated nucleic acids thatencode caninized murine anti-canine IL-4R_(a) antibodies or portionsthereof. In related embodiments such antibodies or antigen bindingfragments can be used for the preparation of a medicament to treatatopic dermatitis in a canine subject. Alternatively, or in conjunction,the present invention provides for the use of any of the antibodies orantibody fragments of the present invention for diagnostic use. In yetadditional embodiments, a kit is provided comprising any of thecaninized antibodies or antigen binding fragments disclosed herein.

In yet additional embodiments, an expression vector is providedcomprising an isolated nucleic acid encoding any of the caninized murineanti-canine IL-4R_(a) antibodies or antigen binding fragments of theinvention. The invention also relates to a host cell comprising any ofthe expression vectors described herein. In particular embodiments,these nucleic acids, expression vectors or polypeptides of the inventionare useful in methods of making an antibody. The present inventionfurther provides peptides (including isolated antigenic peptides) thatconsist of 80 or fewer amino acid residues that comprise the amino acidsequence of SEQ ID NO: 125, or SEQ ID NO: 126, or SEQ ID NO: 127, or SEQID NO: 128, or SEQ ID NO: 154, or SEQ ID NO: 155, or SEQ ID NO: 156, orSEQ ID NO: 157, or SEQ ID NO: 158, or SEQ ID NO: 159, or SEQ ID NO: 160,or SEQ ID NO: 161, or SEQ ID NO: 162. In related embodiments, thepeptides (including isolated antigenic peptides) consist of 60 or feweramino acid residues that comprise the amino acid sequence of SEQ ID NO:125, or SEQ ID NO: 126, or SEQ ID NO: 127, or SEQ ID NO: 128, or SEQ IDNO: 154, or SEQ ID NO: 155, or SEQ ID NO: 156, or SEQ ID NO: 157, or SEQID NO: 158, or SEQ ID NO: 159, or SEQ ID NO: 160, or SEQ ID NO: 161, orSEQ ID NO: 162. In related embodiments, the peptides (including isolatedantigenic peptides) consist of 10 to 45 amino acid residues thatcomprise the amino acid sequence of SEQ ID NO: 125, or SEQ ID NO: 126,or SEQ ID NO: 127, or SEQ ID NO: 128, or SEQ ID NO: 154, or SEQ ID NO:155, or SEQ ID NO: 156, or SEQ ID NO: 157, or SEQ ID NO: 158, or SEQ IDNO: 159, or SEQ ID NO: 160, or SEQ ID NO: 161, or SEQ ID NO: 162. In yetother embodiments the peptides (including isolated antigenic peptides)consist of 5 to 25 amino acid residues from the, or that comprise theamino acid sequence of SEQ ID NO: 125, or SEQ ID NO: 126, or SEQ ID NO:127, or SEQ ID NO: 128, or SEQ ID NO: 154, or SEQ ID NO: 155, or SEQ IDNO: 156, or SEQ ID NO: 157, or SEQ ID NO: 158, or SEQ ID NO: 159, or SEQID NO: 160, or SEQ ID NO: 161, or SEQ ID NO: 162.

The present invention further provides antigenic peptides (includingisolated peptides) that consist of 80 or fewer amino acid residues thatcomprise an amino acid sequence that is 80%, 85%, 90%, 95% or 100%identical with the amino acid sequence of SEQ ID NO: 125, or SEQ ID NO:126, or SEQ ID NO: 127, or SEQ ID NO: 128, or SEQ ID NO: 154, or SEQ IDNO: 155, or SEQ ID NO: 156, or SEQ ID NO: 157, or SEQ ID NO: 158, or SEQID NO: 159, or SEQ ID NO: 160, or SEQ ID NO: 161, or SEQ ID NO: 162 andbinds to an isolated mammalian antibody or antigen binding fragmentthereof the present invention. In related embodiments, the antigenicpeptides (including isolated antigenic peptides) consist of 60 or feweramino acid residues that comprise an amino acid sequence that is 80%,85%, 90%, 95% or 100% identical with the amino acid sequence of SEQ IDNO: 125, or SEQ ID NO: 126, or SEQ ID NO: 127, or SEQ ID NO: 128, or SEQID NO: 154, or SEQ ID NO: 155, or SEQ ID NO: 156, or SEQ ID NO: 157, orSEQ ID NO: 158, or SEQ ID NO: 159, or SEQ ID NO: 160, or SEQ ID NO: 161,or SEQ ID NO: 162 and binds to an isolated mammalian antibody or antigenbinding fragment thereof. In other embodiments the peptides consist of 5to 25 amino acid residues from the, or that comprise an amino acidsequence that is 80%, 85%, 90%, 95% or 100% identical with the aminoacid sequence of SEQ ID NO: 125, or SEQ ID NO: 126, or SEQ ID NO: 127,or SEQ ID NO: 128, or SEQ ID NO: 154, or SEQ ID NO: 155, or SEQ ID NO:156, or SEQ ID NO: 157, or SEQ ID NO: 158, or SEQ ID NO: 159, or SEQ IDNO: 160, or SEQ ID NO: 161, or SEQ ID NO: 162 and binds to an isolatedmammalian antibody or antigen binding fragment thereof. In particularembodiments the mammalian antibody comprises the CDRs of 4D8. In otherembodiments the mammalian antibody comprises the CDRs of 11H2. In yetother embodiments the mammalian antibody comprises the CDRs of 4H3. Instill other embodiments the mammalian antibody comprises the CDRs of11B6. In yet other embodiments the mammalian antibody comprises the CDRsof 2E2. In still other embodiments the mammalian antibody comprises theCDRs of 6C12.

The present invention further provides fusion proteins that comprise anyof the aforesaid peptides. In a particular embodiment, the fusionprotein comprises such an antigenic peptide and an Fc region of anon-canine mammalian IgG antibody. In a more particular embodiment thefusion protein comprises an Fc region of a non-canine mammalian IgGantibody. In certain embodiments the non-canine mammalian IgG antibodyis a murine IgG. In alternative embodiments the non-canine mammalian IgGantibody is a human IgG. In other embodiments the non-canine mammalianIgG antibody is an equine IgG. In still other embodiments the non-caninemammalian IgG antibody is a porcine IgG. In yet other embodiments thenon-canine mammalian IgG antibody is a bovine IgG.

In particular embodiments the non-canine mammalian IgG antibody is anIgG1. In other embodiments the non-canine mammalian IgG antibody is anIgG2a. In still other embodiments the non-canine mammalian IgG antibodyis an IgG3. In yet other embodiments the non-canine mammalian IgGantibody is an IgG4. In other embodiments the fusion protein comprisesany of the aforesaid antigenic peptides and maltose-binding protein. Inyet other embodiments, the fusion protein comprises any of the aforesaidantigenic peptides and beta-galactosidase. In still other embodimentsthe fusion protein comprises any of the aforesaid antigenic peptides andglutathione S-transferase. In yet other embodiments, the fusion proteincomprises any of the aforesaid antigenic peptides and thioredoxin. Instill other embodiments the fusion protein comprises any of theaforesaid antigenic peptides and Gro EL. In yet other embodiments thefusion protein comprises any of the aforesaid antigenic peptides andNusA.

The present invention further provides nucleic acids (including isolatednucleic acids) that encode the antigenic peptides and the correspondingfusion proteins of the present invention. The present invention alsoprovides expression vectors that comprise these nucleic acids and hostcells that comprise one or more expression vectors of the presentinvention.

In addition, the present invention includes pharmaceutical compositionscomprising anti-canine IL-4R_(a) antibodies or antigen binding fragmentsthereof of the present invention, antigenic peptides (including isolatedantigenic peptides) from canine IL-4R_(a), fusion proteins comprisingthe antigenic peptides from canine IL-4R_(a) of the present invention,nucleic acids (including isolated nucleic acids) encoding the antigenicfragments and/or fusion proteins of the present invention, theexpression vectors comprising such nucleic acids, or any combinationthereof, and a pharmaceutically acceptable carrier or diluent.

In addition, the present invention provides methods of negativelyattenuating the activity of IL-4 and/or IL-13 comprising administeringto an animal subject in need thereof a therapeutically effective amountof such pharmaceutical compositions. In certain embodiments the methodis used for the treatment of atopic dermatitis in a canine.

These and other aspects of the present invention will be betterappreciated by reference to the following Brief Description of theDrawings and the Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the reactivity of purified mouse anti-canine IL-4R_(α)monoclonal antibodies (mAbs) against the extracellular domain of canineIL-4R_(a). Various mouse mAbs were tested for their binding to theextracellular domain of canine IL-4R_(a) by ELISA. The mAbs tested aredesignated as: 1A3(●), 1A9(▪), 1B12 (▴), 10C12(▾), 10F2(♦), 10E10(●), 10G8(▪), 11B6(▴), 11D3(▾) and the control antibody(♦). The abscissadepicts the log concentration of the mAB (nM) being added, the ordinatedepicts the optical density obtained by the ELISA.

FIG. 2A shows the dose response curve for the binding of canine IL-4 tocanine IL-4R_(a) expressed on the surface of CHO cells, using acell-based CHO-cIL-4R_(a) binding assay. The abscissa depicts the logconcentration of IL-4 being added, the ordinate depicts the meanfluorescence intensity (MFI) employing FACS.

FIG. 2B depicts the dose response curves for CHO-cIL-4R_(a) by the mouseanti-canine IL-4R_(a) monoclonal antibodies (mAbs): 11B6(●), 4D8(▪),4H3(▴), 2E2(▾), 11H2(♦), and 6C12(◯). The abscissa depicts the logconcentration of the mAb (nM) being added, the ordinate depicts the meanfluorescence intensity (MFI) employing FACS. The half maximal effectiveconcentrations (EC50) for each of the antibodies is provided in Table 2below.

FIGS. 3A and 3B show the results of the addition of successively dilutedindividual mouse anti-canine IL-4R_(a) monoclonal antibodies (mAbs) onthe binding of IL-4 with the cell-based CHO-cIL-4R_(a). FIG. 3A depictsthe concentration-dependent ability of the monoclonal antibodies11B6(♦), 4D8(▪), 4H3(▴), 2E2(▾), and 11H2(♦) to individually block thebinding of IL-4 with the cell-based CHO-cIL-4R_(a). FIG. 3B depicts theconcentration-dependent ability of monoclonal antibodies 11H2(♦), and6C12(▪) to individually block the binding of IL-4 with the cell-basedCHO-cIL-4R_(a). The abscissa depicts the log concentration of the mAb(nM) being added, the ordinate depicts the mean fluorescence intensity(MFI) employing FACS.

FIG. 4 depicts the binding of chimeric and caninized monoclonalantibodies to canine IL-4R_(a) as evaluated by ELISA. The dose-dependentreactivity of caninized monoclonal antibodies against canine IL-4receptor alpha chain is as follows: 4H3 M-C(●); 2G9 M-C(⋄); c4H3 H1-L1(▪); c4H3 H2-L2(▴); c4H3 H3-L3 (∘).

DETAILED DESCRIPTION

A variety of approaches for treatment of human AD are now underinvestigation in many clinical trials [reviewed in Malajian et al., Newpathogenic and therapeutic paradigms in atopic dermatitis Cytokine,(2014)]. Some of these approaches aim to interfere with one or more ofthe signaling molecules/events leading to the development and activationof Th2 cells. One line of investigation in this area encompassesapproaches for blockade of the actions of key interleukin drivers of theTh2 pathway. Based on the observations that AD is largely a Th2dominated disease and the accumulating data supporting a key role forthe combined actions of both IL-4 and IL-13 as key drivers of Th2 celldevelopment, and based on the data indicating that IL-4 receptor α chainis a requisite receptor for signaling from both cytokines, the presentinvention describes the generation and characterization of monoclonalantibodies that block the binding of canine IL-4 and canine IL-13 to thetype-I and type II IL-4 receptors and subsequently inhibit the signalingfrom both canine IL-4 and IL-13. These antibodies have utilities intreatment of atopic dermatitis and other diseases in companion animalsas disclosed herein.

Abbreviations

Throughout the detailed description and examples of the invention thefollowing abbreviations will be used:

-   ADCC Antibody-dependent cellular cytotoxicity-   CDC Complement-dependent cyotoxicity-   CDR Complementarity determining region in the immunoglobulin    variable regions, defined using the Kabat numbering system-   CHO Chinese hamster ovary-   EC50 concentration resulting in 50% efficacy or binding-   ELISA Enzyme-linked immunosorbant assay-   FR Antibody framework region: the immunoglobulin variable regions    excluding the CDR regions.-   HRP Horseradish peroxidase-   IFN interferon-   IC50 concentration resulting in 50% inhibition-   IgG Immunoglobulin G-   Kabat An immunoglobulin alignment and numbering system pioneered by    Elvin A. Kabat [Sequences of Proteins of Immunological Interest, 5th    Ed. Public Health Service, National Institutes of Health, Bethesda,    Md. (1991)]-   mAb Monoclonal antibody (also Mab or MAb)-   MES 2-(N-morpholino)ethanesulfonic acid-   MOA Mechanism of action-   NHS Normal human serum-   PCR Polymerase chain reaction-   PK Pharmacokinetics-   SEB Staphylococcus Enterotoxin B-   TT Tetanus toxoid-   V region The segment of IgG chains which is variable in sequence    between different antibodies. It extends to Kabat residue 109 in the    light chain and 113 in the heavy chain.-   VH Immunoglobulin heavy chain variable region-   VL Immunoglobulin light chain variable region-   VK Immunoglobulin kappa light chain variable region

Definitions

So that the invention may be more readily understood, certain technicaland scientific terms are specifically defined below. Unless specificallydefined elsewhere in this document, all other technical and scientificterms used herein have the meaning commonly understood by one ofordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the,” include their corresponding pluralreferences unless the context clearly dictates otherwise.

“Activation” as it applies to cells or to receptors refers to theactivation or treatment of a cell or receptor with a ligand, unlessindicated otherwise by the context or explicitly. “Ligand” encompassesnatural and synthetic ligands, e.g., cytokines, cytokine variants,analogues, muteins, and binding compounds derived from antibodies.“Ligand” also encompasses small molecules, e.g., peptide mimetics ofcytokines and peptide mimetics of antibodies. “Activation” can refer tocell activation as regulated by internal mechanisms as well as byexternal or environmental factors.

“Activity” of a molecule may describe or refer to the binding of themolecule to a ligand or to a receptor, to catalytic activity; to theability to stimulate gene expression or cell signaling, differentiation,or maturation; to antigenic activity, to the modulation of activities ofother molecules, and the like. “Activity” of a molecule may also referto activity in modulating or maintaining cell-to-cell interactions,e.g., adhesion, or activity in maintaining a structure of a cell, e.g.,cell membranes or cytoskeleton. “Activity” can also mean specificactivity, e.g., [catalytic activity]/[mg protein], or [immunologicalactivity]/[mg protein], concentration in a biological compartment, orthe like. “Activity” may refer to modulation of components of the innateor the adaptive immune systems.

“Administration” and “treatment,” as it applies to an animal, e.g., acanine experimental subject, cell, tissue, organ, or biological fluid,refers to contact of an exogenous pharmaceutical, therapeutic,diagnostic agent, or composition to the animal e.g., a canine subject,cell, tissue, organ, or biological fluid. Treatment of a cellencompasses contact of a reagent to the cell, as well as contact of areagent to a fluid, where the fluid is in contact with the cell.“Administration” and “treatment” also means in vitro and ex vivotreatments, e.g., of a cell, by a reagent, diagnostic, binding compound,or by another cell. The term “subject” includes any organism, preferablyan animal, more preferably a mammal (e.g., canine, feline, or human) andmost preferably a canine.

As used herein, a “substitution of an amino acid residue” with anotheramino acid residue in an amino acid sequence of an antibody for example,is equivalent to “replacing an amino acid residue” with another aminoacid residue and denotes that a particular amino acid residue at aspecific position in the amino acid sequence has been replaced by (orsubstituted for) by a different amino acid residue. Such substitutionscan be particularly designed i.e., purposefully replacing an alaninewith a serine at a specific position in the amino acid sequence by e.g.,recombinant DNA technology. Alternatively, a particular amino acidresidue or string of amino acid residues of an antibody can be replacedby one or more amino acid residues through more natural selectionprocesses e.g., based on the ability of the antibody produced by a cellto bind to a given region on that antigen, e.g., one containing anepitope or a portion thereof, and/or for the antibody to comprise aparticular CDR that retains the same canonical structure as the CDR itis replacing. Such substitutions/replacements can lead to “variant” CDRsand/or variant antibodies.

“Treat” or “treating” means to administer a therapeutic agent, such as acomposition containing any of the antibodies or antigen bindingfragments of the present invention, internally or externally to a caninesubject or patient having one or more disease symptoms, or beingsuspected of having a disease, for which the agent has therapeuticactivity.

Typically, the agent is administered in an amount effective to alleviateand/or ameliorate one or more disease symptoms in the treated subject orpopulation, whether by inducing the regression of or inhibiting theprogression of such symptom(s) by any clinically measurable degree. Theamount of a therapeutic agent that is effective to alleviate anyparticular disease symptom (also referred to as the “therapeuticallyeffective amount”) may vary according to factors such as the diseasestate, age, and weight of the patient (e.g., canine), and the ability ofthe pharmaceutical composition to elicit a desired response in thesubject. Whether a disease symptom has been alleviated or amelioratedcan be assessed by any clinical measurement typically used byveterinarians or other skilled healthcare providers to assess theseverity or progression status of that symptom. While an embodiment ofthe present invention (e.g., a treatment method or article ofmanufacture) may not be effective in alleviating the target diseasesymptom(s) in every subject, it should alleviate the target diseasesymptom(s) in a statistically significant number of subjects asdetermined by any statistical test known in the art such as theStudent's t-test, the chi²-test, the U-test according to Mann andWhitney, the Kruskal-Wallis test (H-test), Jonckheere-Terpstra-test andthe Wilcoxon-test.

“Treatment,” as it applies to a human, veterinary (e.g., canine) orresearch subject, refers to therapeutic treatment, as well as researchand diagnostic applications. “Treatment” as it applies to a human,veterinary (e.g., canine), or research subject, or cell, tissue, ororgan, encompasses contact of the antibodies or antigen bindingfragments of the present invention to a canine or other animal subject,a cell, tissue, physiological compartment, or physiological fluid.

As used herein, the term “canine” includes all domestic dogs, Canislupus familiaris or Canis familiaris, unless otherwise indicated.

As used herein, the term “feline” refers to any member of the Felidaefamily. Members of this family include wild, zoo, and domestic members,such as any member of the subfamilies Felinae, e.g., cats, lions,tigers, pumas, jaguars, leopards, snow leopards, panthers, NorthAmerican mountain lions, cheetahs, lynx, bobcats, caracals or any crossbreeds thereof. Cats also include domestic cats, pure-bred and/ormongrel companion cats, show cats, laboratory cats, cloned cats, andwild or feral cats.

As used herein the term “canine frame” refers to the amino acid sequenceof the heavy chain and light chain of a canine antibody other than thehypervariable region residues defined herein as CDR residues. Withregard to a caninized antibody, in the majority of embodiments the aminoacid sequences of the native canine CDRs are replaced with thecorresponding foreign CDRs (e.g., those from a mouse antibody) in bothchains. Optionally the heavy and/or light chains of the canine antibodymay contain some foreign non-CDR residues, e.g., so as to preserve theconformation of the foreign CDRs within the canine antibody, and/or tomodify the Fc function, as exemplified below.

Canine IL-4R_(a) has been found to comprise the amino acid sequence ofSEQ ID NO: 2 [SEQ ID NO: 4, without the signal sequence]. In a specificembodiment canine IL-4R_(a) is encoded by a nucleic acid that comprisesthe nucleotide sequence of SEQ ID NO: 1 [SEQ ID NO: 3, without thesignal sequence]. Canine IL-4R_(a) sequences may differ by having, forexample, conserved variations in non-conserved regions, but the canineIL-4R_(a) will have substantially the same biological function as thecanine IL-4R_(a) comprising the amino acid sequence of SEQ ID NO: 2 [SEQID NO: 4, without the signal sequence].

The cytokines IL-4 and IL-13 have been implicated in the pathogenesis ofa variety of allergic diseases in humans and animals, including asthmaand atopic dermatitis. Because the IL-4 receptor α chain is a requisitereceptor for the signaling from either of these cytokines, the presentinvention describes the generation and characterization of monoclonalantibodies that block the binding of canine IL-4 and canine IL-13 toIL-4R_(a) and thereby inhibits the signaling from both canine IL-4 andIL-13. These antibodies therefore have utility in treatment of atopicdermatitis and other diseases in companion animals as disclosed herein.In addition, a biological function of canine IL-4R_(a) may be having,for example, an epitope in the extracellular domain that is specificallybound by an antibody of the instant disclosure.

A particular canine IL-4R_(α) amino acid sequence will generally be atleast 90% identical to the canine IL-4R_(α) comprising the amino acidsequence of SEQ ID NO: 4. In certain cases, a canine IL-4R_(α) may be atleast 95%, or even at least 96%, 97%, 98% or 99% identical to the canineIL-4R_(α) comprising the amino acid sequence of SEQ ID NO: 4. In certainembodiments, a canine IL-4R_(α) amino acid sequence will display no morethan 10 amino acid differences from the canine IL-4R_(α) comprising theamino acid sequence of SEQ ID NO: 4. In certain embodiments, the canineIL-4R_(α) amino acid sequence may display no more than 5, or even nomore than 4, 3, 2, or 1 amino acid difference from the canine IL-4R_(α)comprising the amino acid sequence of SEQ ID NO: 4. Percent identity canbe determined as described herein below.

The term “immune response” refers to the action of, for example,lymphocytes, antigen presenting cells, phagocytic cells, granulocytes,and soluble macromolecules produced by the above cells or the liver(including antibodies, cytokines, and complement) that results inselective damage to, destruction of, or elimination from the mammalianbody (e.g., canine body) of cancerous cells, cells or tissues infectedwith pathogens, or invading pathogens.

Anti-Canine IL-4R_(α) Antibodies

The present invention provides isolated antibodies (particularly murineanti-canine IL-4R_(a) antibodies and caninized antibodies thereof) orantigen binding fragments thereof that bind canine IL-4R_(α) and uses ofsuch antibodies or fragments thereof. In specific embodiments murineanti-canine IL-4R_(α) CDRs from murine anti-canine IL-4R_(α) antibodiesare provided that have been shown to both bind canine IL-4R_(α) and toblock the binding of canine IL-4R_(α) to one or more of its ligands,canine IL-4 or IL-13. These CDRs can be inserted into a modified canineframe of a canine antibody to generate a caninized murine anti-canineIL-4R_(α) antibody.

As used herein, an “anti-canine IL-4R_(α) antibody” refers to anantibody that was raised against canine IL-4R_(α) (e.g., in a mammalsuch as a mouse or rabbit) and that specifically binds to canineIL-4R_(α). An antibody that “specifically binds to canine IL-4R_(α),”and in particular canine IL-4R_(α), or an antibody that “specificallybinds to a polypeptide comprising the amino acid sequence of canineIL-4R_(α)”, is an antibody that exhibits preferential binding to canineIL-4R_(α) as compared to other antigens, but this specificity does notrequire absolute binding specificity. An anti-canine IL-4R_(α) antibodyis considered “specific” for canine IL-4R_(α) if its binding isdeterminative of the presence of canine IL-4R_(α) in a sample, or if itis capable of altering the activity of canine IL-4R_(α) without undulyinterfering with the activity of other molecules in a canine sample,e.g. without producing undesired results such as false positives in adiagnostic context or side effects in a therapeutic context. The degreeof specificity necessary for an anti-canine IL-4R_(α) antibody maydepend on the intended use of the antibody, and at any rate is definedby its suitability for use for an intended purpose. The antibody, orbinding compound derived from the antigen-binding site of an antibody,of the contemplated method binds to its antigen, or a variant or muteinthereof, with an affinity that is at least two-fold greater, preferablyat least ten-times greater, more preferably at least 20-times greater,and most preferably at least 100-times greater than the affinity withany other antigen.

As used herein, an antibody is said to bind specifically to apolypeptide comprising a given antigen sequence (in this case a portionof the amino acid sequence of canine IL-4R_(α)) if it binds topolypeptides comprising the portion of the amino acid sequence of canineIL-4R_(α), but does not bind to other canine proteins lacking thatportion of the sequence of canine IL-4R_(α). For example, an antibodythat specifically binds to a polypeptide comprising canine IL-4R_(α),may bind to a FLAG®-tagged form of canine IL-4R_(α), but will not bindto other FLAG®-tagged canine proteins. An antibody, or binding compoundderived from the antigen-binding site of an antibody, binds to itscanine antigen, or a variant or mutein thereof, “with specificity” whenit has an affinity for that canine antigen or a variant or muteinthereof which is at least ten-times greater, more preferably at least20-times greater, and even more preferably at least 100-times greaterthan its affinity for any other canine antigen tested.

As used herein, the term “antibody” refers to any form of antibody thatexhibits the desired biological activity. Thus, it is used in thebroadest sense and specifically covers, but is not limited to,monoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), canonized antibodies, fully canine antibodies, chimericantibodies and camelized single domain antibodies. “Parental antibodies”are antibodies obtained by exposure of an immune system to an antigenprior to modification of the antibodies for an intended use, such ascaninization of an antibody for use as a canine therapeutic antibody.

As used herein, unless otherwise indicated, “antibody fragment” or“antigen binding fragment” refers to antigen binding fragments ofantibodies, i.e. antibody fragments that retain the ability to bindspecifically to the antigen bound by the full-length antibody, e.g.fragments that retain one or more CDR regions. Examples of antigenbinding fragments include, but are not limited to, Fab, Fab′, F(ab′)₂,and Fv fragments; diabodies; linear antibodies; single-chain antibodymolecules, e.g., sc-Fv; nanobodies and multispecific antibodies formedfrom antibody fragments.

A “Fab fragment” is comprised of one light chain and the C_(H)1 andvariable regions of one heavy chain. The heavy chain of a Fab moleculecannot form a disulfide bond with another heavy chain molecule. A “Fabfragment” can be the product of papain cleavage of an antibody.

A “fragment crystallizable” (“Fc”) region contains two heavy chainfragments comprising the C_(H)3 and C_(H)2 domains of an antibody. Thetwo heavy chain fragments are held together by two or more disulfidebonds and by hydrophobic interactions of the C_(H)3 domains.

A “Fab′ fragment” contains one light chain and a portion or fragment ofone heavy chain that contains the V_(H) domain and the C_(H)1 domain andalso the region between the C_(H)1 and C_(H)2 domains, such that aninterchain disulfide bond can be formed between the two heavy chains oftwo Fab′ fragments to form a F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 andC_(H)2 domains, such that an interchain disulfide bond is formed betweenthe two heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains. An “F(ab′)₂ fragment” can be the product of pepsincleavage of an antibody.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

The term “single-chain Fv” or “scFv” antibody refers to antibodyfragments comprising the V_(H) and V_(L) domains of an antibody, whereinthese domains are present in a single polypeptide chain. Generally, theFv polypeptide further comprises a polypeptide linker between the V_(H)and V_(L) domains which enables the scFv to form the desired structurefor antigen binding. [See, Pluckthun, THE PHARMACOLOGY OF MONOCLONALANTIBODIES, vol. 113 Rosenburg and Moore eds., Springer-Verlag, NewYork, pp. 269-315 (1994); WO 88/01649; and U.S. Pat. Nos. 4,946,778 and5,260,203.]

As used herein, the term “canonical structure” refers to the localconformation that can be adopted by each of the hypervariable regions ofthe heavy and light chain of an antibody within the framework that theyreside. For each hypervariable region, there are a small number ofcanonical structures (generally denoted by simple integers such as 1 or2 etc.), which can be predicted with great accuracy from the amino acidsequences of the corresponding hypervariable region [particularly withinthe context of the amino acid sequence of its framework, as providedbelow for the corresponding anti-canine IL-4R_(a) variable domains (see,Table 3 below)]. These canonical structures can be determinativeregarding whether a modification of the amino acid sequence of a givenCDR will result in the retention or loss of the ability to bind to itsantigen binding partner [See, Chothia and Lesk, Canonical Structures forthe hypervariable regions of immunoglobulins, J. Mol. Biol.196:901-917(1987); Chothia et al., Conformation of immunoglobulinhypervaribale regions, Nature, 34:877-883(1989); and Al-Lazikani et al.,Standard Conformations for the canonical structures of immunoglobulins,J. Mol. Biol. 273:927-948 (1997)].

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody maytarget the same or different antigens.

A “bivalent antibody” comprises two antigen binding sites. In someinstances, the two binding sites have the same antigen specificities.However, bivalent antibodies may be bispecific (see below).

In certain embodiments, monoclonal antibodies herein also includecamelized single domain antibodies. [See, e.g., Muyldermans et al.,Trends Biochem. Sci. 26:230 (2001); Reichmann et al., J. Immunol.Methods 231:25 (1999); WO 94/04678; WO 94/25591; U.S. Pat. No.6,005,079]. In one embodiment, the present invention provides singledomain antibodies comprising two V_(H) domains with modifications suchthat single domain antibodies are formed.

As used herein, the term “diabodies” refers to small antibody fragmentswith two antigen-binding sites, which fragments comprise a heavy chainvariable domain (V_(H)) connected to a light chain variable domain(V_(L)) in the same polypeptide chain (V_(H)-V_(L) or V_(L)-V_(H)). Byusing a linker that is too short to allow pairing between the twodomains on the same chain, the domains are forced to pair with thecomplementary domains of another chain and create two antigen-bindingsites. [See, EP 0 404 097 B1; WO 93/11161; and Holliger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993)]. For a review of engineeredantibody variants [generally see Holliger and Hudson Nat. Biotechnol.23:1126-1136 (2005)].

Typically, an antibody or antigen binding fragment of the inventionretains at least 10% of its canine IL-4R_(a) binding activity (whencompared to the parental antibody) when that activity is expressed on amolar basis. Preferably, an antibody or antigen binding fragment of theinvention retains at least 20%, 50%, 70%, 80%, 90%, 95% or 100% or moreof the canine IL-4R_(a) binding affinity as the parental antibody. It isalso intended that an antibody or antigen binding fragment of theinvention can include conservative or non-conservative amino acidsubstitutions (referred to as “conservative variants” or “functionconserved variants” of the antibody) that do not substantially alter itsbiologic activity.

“Isolated antibody” refers to the purification status and in suchcontext means the molecule is substantially free of other biologicalmolecules such as nucleic acids, proteins, lipids, carbohydrates, orother material such as cellular debris and growth media. Generally, theterm “isolated” is not intended to refer to a complete absence of suchmaterial or to an absence of water, buffers, or salts, unless they arepresent in amounts that substantially interfere with experimental ortherapeutic use of the binding compound as described herein.

As used herein, a “chimeric antibody” is an antibody having the variabledomain from a first antibody and the constant domain from a secondantibody, where the first and second antibodies are from differentspecies. [U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl.Acad. Sci. USA 81: 6851-6855 (1984)]. Typically the variable domains areobtained from an antibody from an experimental animal (the “parentalantibody”), such as a rodent, and the constant domain sequences areobtained from the animal subject antibodies, e.g., human or canine sothat the resulting chimeric antibody will be less likely to elicit anadverse immune response in a canine or human subject respectively, thanthe parental (e.g., rodent) antibody.

As used herein, the term “caninized antibody” refers to forms ofantibodies that contain sequences from both canine and non-canine (e.g.,murine) antibodies. In general, the caninized antibody will comprisesubstantially all of at least one or more typically, two variabledomains in which all or substantially all of the hypervariable loopscorrespond to those of a non-canine immunoglobulin (e.g., comprising 6murine anti-canine IL-4R_(a) CDRs as exemplified below), and all orsubstantially all of the framework (FR) regions (and typically all orsubstantially all of the remaining frame) are those of a canineimmunoglobulin sequence. As exemplified herein, a caninized antibodycomprises both the three heavy chain CDRs and the three light chain CDRSfrom a murine anti-canine IL-4R_(a) antibody together with a canineframe or a modified canine frame. A modified canine frame comprises oneor more amino acids changes as exemplified herein that further optimizethe effectiveness of the caninized antibody, e.g., to increase itsbinding to canine IL-4R_(a) and/or its ability to block the binding ofcanine IL-4 and/or canine IL-13 to the type-I and/or type II IL-4receptors.

The term “fully canine antibody” refers to an antibody that comprisescanine immunoglobulin protein sequences only. A fully canine antibodymay contain murine carbohydrate chains if produced in a mouse, in amouse cell, or in a hybridoma derived from a mouse cell. Similarly,“mouse antibody” refers to an antibody that comprises mouseimmunoglobulin sequences only. Alternatively, a fully canine antibodymay contain rat carbohydrate chains if produced in a rat, in a rat cell,or in a hybridoma derived from a rat cell. Similarly, “rat antibody”refers to an antibody that comprises rat immunoglobulin sequences only.

There are four known IgG heavy chain subtypes of dog IgG and they arereferred to as IgG-A, IgG-B, IgG-C, and IgG-D. The two known light chainsubtypes are referred to as lambda and kappa.

The variable regions of each light/heavy chain pair form the antibodybinding site. Thus, in general, an intact antibody has two bindingsites. Except in bifunctional or bispecific antibodies, the two bindingsites are, in general, the same.

Typically, the variable domains of both the heavy and light chainscomprise three hypervariable regions, also called complementaritydetermining regions (CDRs), located within relatively conservedframework regions (FR). The CDRs are usually aligned by the frameworkregions, enabling binding to a specific epitope. In general, fromN-terminal to C-terminal, both light and heavy chains variable domainscomprise FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment ofamino acids to each domain is, generally, in accordance with thedefinitions of Sequences of Proteins of Immunological Interest, Kabat,et al.; National Institutes of Health, Bethesda, Md.; 5^(th) ed.; NIHPubl. No. 91-3242 (1991); Kabat, Adv. Prot. Chem. 32:1-75 (1978); Kabat,et al., J. Biol. Chem. 252:6609-6616 (1977); Chothia, et al., J. Mol.Biol. 196:901-917 (1987) or Chothia, et al., Nature 342:878-883 (1989)].

As used herein, the term “hypervariable region” refers to the amino acidresidues of an antibody that are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a“complementarity determining region” or “CDR” (i.e. CDRL1, CDRL2 andCDRL3 in the light chain variable domain and CDRH1, CDRH2 and CDRH3 inthe heavy chain variable domain). [See Kabat et al. Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991), defining the CDRregions of an antibody by sequence; see also Chothia and Lesk, J. Mol.Biol. 196: 901-917 (1987) defining the CDR regions of an antibody bystructure]. As used herein, the term “framework” or “FR” residues refersto those variable domain residues other than the hypervariable regionresidues defined herein as CDR residues.

Besides binding and activating of canine immune cells, a canine orcaninized antibody against IL-4R_(a) optimally has two attributes:

-   -   1. Lack of effector functions such as antibody-dependent        cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC),        and    -   2. be readily purified on a large scale using industry standard        technologies such as that based on protein A chromatography.

None of the naturally occurring canine IgG isotypes satisfy bothcriteria. For example, IgG-B can be purified using protein A, but hashigh level of ADCC activity. On the other hand, IgG-A binds weakly toprotein A, but displays undesirable ADCC activity. Moreover, neitherIgG-C nor IgG-D can be purified on protein A columns, although IgG-Ddisplay no ADCC activity. (IgG-C has considerable ADCC activity). Oneway the present invention overcomes this difficulty is by providingmutant canine IgG-B antibodies specific to IL-4R_(a); such antibodieslack effector functions such as ADCC and can be easily of purified usingindustry standard protein A chromatography.

“Homology” refers to sequence similarity between two polynucleotidesequences or between two polypeptide sequences when they are optimallyaligned. When a position in both of the two compared sequences isoccupied by the same base or amino acid monomer subunit, e.g., if aposition in each of two DNA molecules is occupied by adenine, then themolecules are homologous at that position. The percent of homology isthe number of homologous positions shared by the two sequences dividedby the total number of positions compared ×100. For example, if 6 of 10of the positions in two sequences are matched or homologous when thesequences are optimally aligned then the two sequences are 60%homologous. Generally, the comparison is made when two sequences arealigned to give maximum percent homology.

“Isolated nucleic acid molecule” means a DNA or RNA of genomic, mRNA,cDNA, or synthetic origin or some combination thereof which is notassociated with all or a portion of a polynucleotide in which theisolated polynucleotide is found in nature, or is linked to apolynucleotide to which it is not linked in nature. For purposes of thisdisclosure, it should be understood that “a nucleic acid moleculecomprising” a particular nucleotide sequence does not encompass intactchromosomes. Isolated nucleic acid molecules “comprising” specifiednucleic acid sequences may include, in addition to the specifiedsequences, coding sequences for up to ten or even up to twenty or moreother proteins or portions or fragments thereof, or may include operablylinked regulatory sequences that control expression of the coding regionof the recited nucleic acid sequences, and/or may include vectorsequences.

The phrase “control sequences” refers to DNA sequences necessary for theexpression of an operably linked coding sequence in a particular hostorganism. The control sequences that are suitable for prokaryotes, forexample, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to use promoters,polyadenylation signals, and enhancers.

A nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. Generally, “operably linked”means that the DNA sequences being linked are contiguous, and, in thecase of a secretory leader, contiguous and in reading phase. However,enhancers do not have to be contiguous. Linking is accomplished byligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice.

As used herein, the expressions “cell,” “cell line,” and “cell culture”are used interchangeably and all such designations include progeny.Thus, the words “transformants” and “transformed cells” include theprimary subject cell and cultures derived therefrom without regard forthe number of transfers. It is also understood that not all progeny willhave precisely identical DNA content, due to deliberate or inadvertentmutations. Mutant progeny that have the same function or biologicalactivity as screened for in the originally transformed cell areincluded. Where distinct designations are intended, it will be clearfrom the context.

As used herein, “germline sequence” refers to a sequence of unrearrangedimmunoglobulin DNA sequences. Any suitable source of unrearrangedimmunoglobulin sequences may be used. Human germline sequences may beobtained, for example, from JOINSOLVER® germline databases on thewebsite for the National Institute of Arthritis and Musculoskeletal andSkin Diseases of the United States National Institutes of Health. Mousegermline sequences may be obtained, for example, as described inGiudicelli et al. [Nucleic Acids Res. 33:D256-D261 (2005)].

Properties of Murine Anti-Canine IL-4R_(α) and Caninized MurineAnti-Canine IL-4R_(α) Antibodies

The present invention provides isolated murine anti-canine IL-4R_(α)antibodies and caninized antibodies thereof, methods of use of theantibodies or antigen binding fragments thereof in the treatment ofdisease e.g., the treatment of atopic dermatitis in canines. In canine,there are four IgG heavy chains referred to as A, B, C, and D. Theseheavy chains represent four different subclasses of dog IgG, which arereferred to as IgGA, IgGB, IgGC and IgGD. Each of the two heavy chainsconsists of one variable domain (VH) and three constant domains referredto as CH-1, CH-2, and CH-3. The CH-1 domain is connected to the CH-2domain via an amino acid sequence referred to as the “hinge” oralternatively as the “hinge region”.

The DNA and amino acid sequences of these four heavy chains were firstidentified by Tang et al. [Vet. Immunol. Immunopathol. 80: 259-270(2001)]. The amino acid and DNA sequences for these heavy chains arealso available from the GenBank data bases. For example, the amino acidsequence of IgGA heavy chain has accession number AAL35301.1, IgGB hasaccession number AAL35302.1, IgGC has accession number AAL35303.1, andIgGD has accession number (AAL35304.1). Canine antibodies also containtwo types of light chains, kappa and lambda. The DNA and amino acidsequence of these light chains can be obtained from GenBank Databases.For example the kappa light chain amino acid sequence has accessionnumber ABY 57289.1 and the lambda light chain has accession number ABY55569.1.

In the present invention, the amino acid sequence for each of the fourcanine IgG Fc fragments is based on the identified boundary of CH1 andCH2 domains as determined by Tang et al, supra. Caninized murineanti-canine IL-4R_(α) antibodies that bind canine IL-4R_(α) include, butare not limited to: antibodies that comprise canine IgG-A, IgG-B, andIgG-D heavy chains and/or canine kappa light chains together with murineanti-canine IL-4R_(α) CDRs. Accordingly, the present invention providesisolated murine anti-canine IL-4R_(α) and/or caninized murineanti-canine IL-4R_(α) antibodies or antigen binding fragments thereofthat bind to canine IL-4R_(α) and block the binding of canine IL-4 andcanine IL-13 to the type-I or type II IL-4 receptors.

The present invention further provides full length canine heavy chainsthat can be matched with corresponding light chains to make a caninizedantibody. Accordingly, the present invention further provides caninizedmurine anti-canine antigen antibodies (including isolated caninizedmurine anti-canine IL-4R_(α) antibodies) and methods of use of theantibodies or antigen binding fragments thereof in the treatment ofdisease e.g., the treatment of atopic dematitis in canines.

The present invention also provides caninized murine anti-canine-IL-4Rαantibodies that comprise a canine fragment crystallizable region (cFcregion) in which the cFc has been genetically modified to augment,decrease, or eliminate one or more effector functions. In one aspect ofthe present invention, the genetically modified cFc decreases oreliminates one or more effector functions. In another aspect of theinvention the genetically modified cFc augments one or more effectorfunction. In certain embodiments, the genetically modified cFc region isa genetically modified canine IgGB Fc region. In another suchembodiment, the genetically modified cFc region is a geneticallymodified canine IgGC Fc region. In a particular embodiment the effectorfunction is antibody-dependent cytotoxicity (ADCC) that is augmented,decreased, or eliminated. In another embodiment the effector function iscomplement-dependent cytotoxicity (CDC) that is augmented, decreased, oreliminated. In yet another embodiment, the cFc region has beengenetically modified to augment, decrease, or eliminate both the ADCCand the CDC.

In order to generate variants of canine IgG that lack effectorfunctions, a number of mutant canine IgGB heavy chains were generated.These variants may include one or more of the following single orcombined substitutions in the Fc portion of the heavy chain amino acidsequence: P4A, D31A, N63A, G64P, T65A, A93G, and P95A. Variant heavychains (i.e., containing such amino acid substitutions) were cloned intoexpression plasmids and transfected into HEK 293 cells along with aplasmid containing the gene encoding a light chain. Intact antibodiesexpressed and purified from HEK 293 cells were evaluated for binding toFc_(γ)RI and C1q to assess their potential for mediation of immuneeffector functions. [see, U.S. provisional patent application62/030,812, filed Jul. 30, 2014, and U.S. provisional patent application62/092,496, filed Dec. 16, 2014, the contents of both of which arehereby incorporated by reference in their entireties.]

The present invention also provides modified canine IgGDs which in placeof its natural IgGD hinge region they comprise a hinge region from:

IgGA: SEQ ID NO: 101 FNECRCTDTPPCPVPEP,; IgGB: SEQ ID NO: 102PKRENGRVPRPPDCPKCPAPEM,; or IgGC: SEQ ID NO: 103 AKECECKCNCNNCPCPGCGL,.

Alternatively, the IgGD hinge region can be genetically modified byreplacing a serine residue with a proline residue, i.e.,PKESTCKCIPCPVPES, SEQ ID NO: 104 (with the proline residue (P)underlined and in bold substituting for the naturally occurring serineresidue). Such modifications can lead to a canine IgGD lacking fab armexchange. The modified canine IgGDs can be constructed using standardmethods of recombinant DNA technology [e.g., Maniatis et al., MolecularCloning, A Laboratory Manual (1982)]. In order to construct thesevariants, the nucleic acids encoding the amino acid sequence of canineIgGD can be modified so that it encodes the modified IgGDs. The modifiednucleic acid sequences are then cloned into expression plasmids forprotein expression.

The antibody or antigen binding fragment thereof that binds canineIL-4R_(α) can comprise one, two, three, four, five, or six of thecomplementarity determining regions (CDRs) of the murine anti-canineantibody as described herein. The one, two, three, four, five, or sixCDRs may be independently selected from the CDR sequences of thoseprovided below. In a further embodiment, the isolated antibody orantigen-binding fragment thereof that binds canine IL-4R_(α) comprises acanine antibody kappa light chain comprising a murine light chain CDR-1,CDR-2 and/or CDR-3 and a canine antibody heavy chain IgG comprising amurine heavy chain CDR-1, CDR-2 and/or CDR-3.

In other embodiments, the invention provides antibodies or antigenbinding fragments thereof that specifically binds IL-4R_(α) and havecanine antibody kappa light chains comprising one to six different CDRscomprising at least 80%, 85%, 90%, 95%, 98% or 99% sequence identitywith the amino acid sequences of SEQ ID NOs: 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, and/or 73 and canine antibody heavy chain IgG comprising one to sixdifferent CDRs comprising at least 80%, 85%, 90%, 95%, 98% or 99%sequence identity with the amino acid sequences of SEQ ID NOs: 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, and/or 100, while still exhibiting the desiredbinding and functional properties. In another embodiment the antibody orantigen binding fragment of the present invention comprises a canineframe comprising a combination of IgG heavy chain sequence with a kappalight chain having one or more of the above-mentioned CDR amino acidsequences with 0, 1, 2, 3, 4, or 5 conservative or non-conservativeamino acid substitutions, while still exhibiting the desired binding andfunctional properties.

Sequence identity refers to the degree to which the amino acids of twopolypeptides are the same at equivalent positions when the two sequencesare optimally aligned. As used herein one amino acid sequence is 100%“identical” to a second amino acid sequence when the amino acid residuesof both sequences are identical. Accordingly, an amino acid sequence is50% “identical” to a second amino acid sequence when 50% of the aminoacid residues of the two amino acid sequences are identical. Thesequence comparison is performed over a contiguous block of amino acidresidues comprised by a given protein, e.g., a protein, or a portion ofthe polypeptide being compared. In a particular embodiment, selecteddeletions or insertions that could otherwise alter the correspondencebetween the two amino acid sequences are taken into account.

Sequence similarity includes identical residues and nonidentical,biochemically related amino acids. Biochemically related amino acidsthat share similar properties and may be interchangeable are discussed

“Conservatively modified variants” or “conservative substitution” refersto substitutions of amino acids in a protein with other amino acidshaving similar characteristics (e.g. charge, side-chain size,hydrophobicity/hydrophilicity, backbone conformation and rigidity,etc.), such that the changes can frequently be made without altering thebiological activity of the protein. Those of skill in this art recognizethat, in general, single amino acid substitutions in non-essentialregions of a polypeptide do not substantially alter biological activity[see, e.g., Watson et al., Molecular Biology of the Gene, TheBenjamin/Cummings Pub. Co., p. 224 (4th Ed.; 1987)]. In addition,substitutions of structurally or functionally similar amino acids areless likely to disrupt biological activity. Exemplary conservativesubstitutions are set forth in Table 1 directly below.

TABLE 1 Exemplary Conservative Amino Acid Substitutions ConservativeOriginal residue substitution Ala (A) Gly; Ser; Arg (R) Lys; His Asn (N)Gln; His Asp (D) Glu; Asn Cys (C) Ser; Ala Gln (Q) Asn Glu (E) Asp; GlnGly (G) Ala His (H) Asn; Gln Ile (I) Leu; Val Leu (L) Ile; Val Lys (K)Arg; His Met (M) Leu; Ile; Tyr Phe (F) Tyr; Met; Leu Pro (P) Ala; GlySer (S) Thr Thr (T) Ser Trp (W) Tyr; Phe Tyr (Y) Trp; Phe Val (V) Ile;Leu

Function-conservative variants of the antibodies of the invention arealso contemplated by the present invention. “Function-conservativevariants,” as used herein, refers to antibodies or fragments in whichone or more amino acid residues have been changed without altering adesired property, such an antigen affinity and/or speficity. Suchvariants include, but are not limited to, replacement of an amino acidwith one having similar properties, such as the conservative amino acidsubstitutions of Table 1 above.

Nucleic Acids

The present invention further comprises the nucleic acids encoding theimmunoglobulin chains of murine anti-canine IL-4R_(α) and/or caninizedmurine anti-canine IL-4R_(α) antibodies and antigen binding fragmentsthereof disclosed herein (see Examples below).

Also included in the present invention are nucleic acids that encodeimmunoglobulin polypeptides comprising amino acid sequences that are atleast about 70% identical, preferably at least about 80% identical, morepreferably at least about 90% identical and most preferably at leastabout 95% identical (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to the aminoacid sequences of the CDRs and antibodies provided herein when thecomparison is performed by a BLAST algorithm wherein the parameters ofthe algorithm are selected to give the largest match between therespective sequences over the entire length of the respective referencesequences. The present invention further provides nucleic acids thatencode immunoglobulin polypeptides comprising amino acid sequences thatare at least about 70% similar, preferably at least about 80% similar,more preferably at least about 90% similar and most preferably at leastabout 95% similar (e.g., 95%, 96%, 97%, 98%, 99%, 100%) to any of thereference amino acid sequences when the comparison is performed with aBLAST algorithm, wherein the parameters of the algorithm are selected togive the largest match between the respective sequences over the entirelength of the respective reference sequences, are also included in thepresent invention.

As used herein, nucleotide and amino acid sequence percent identity canbe determined using C, MacVector (MacVector, Inc. Cary, N.C. 27519),Vector NTI (Informax, Inc. MD), Oxford Molecular Group PLC (1996) andthe Clustal W algorithm with the alignment default parameters, anddefault parameters for identity. These commercially available programscan also be used to determine sequence similarity using the same oranalogous default parameters. Alternatively, an Advanced Blast searchunder the default filter conditions can be used, e.g., using the GCG(Genetics Computer Group, Program Manual for the GCG Package, Version 7,Madison, Wis.) pileup program using the default parameters.

The following references relate to BLAST algorithms often used forsequence analysis: BLAST ALGORITHMS: Altschul, S. F., et al., J. Mol.Biol. 215:403-410 (1990); Gish, W., et al., Nature Genet. 3:266-272(1993); Madden, T. L., et al., Meth. Enzymol. 266:131-141(1996);Altschul, S. F., et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang,J., et al., Genome Res. 7:649-656 (1997); Wootton, J. C., et al.,Comput. Chem. 17:149-163 (1993); Hancock, J. M. et al., Comput. Appl.Biosci. 10:67-70 (1994); ALIGNMENT SCORING SYSTEMS: Dayhoff, M. O., etal., “A model of evolutionary change in proteins.” in Atlas of ProteinSequence and Structure, vol. 5, suppl. 3. M. O. Dayhoff (ed.), pp.345-352, (1978); Natl. Biomed. Res. Found., Washington, D.C.; Schwartz,R. M., et al., “Matrices for detecting distant relationships.” in Atlasof Protein Sequence and Structure, vol. 5, suppl. 3.” (1978), M. O.Dayhoff (ed.), pp. 353-358 (1978), Natl. Biomed. Res. Found.,Washington, D.C.; Altschul, S. F., J. Mol. Biol. 219:555-565 (1991);States, D. J., et al., Methods 3:66-70(1991); Henikoff, S., et al.,Proc. Natl. Acad. Sci. USA 89:10915-10919 (1992); Altschul, S. F., etal., J. Mol. Evol. 36:290-300 (1993); ALIGNMENT STATISTICS: Karlin, S.,et al., Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990); Karlin, S., etal., Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); Dembo, A., et al.,Ann. Prob. 22:2022-2039 (1994); and Altschul, S. F. “Evaluating thestatistical significance of multiple distinct local alignments.” inTheoretical and Computational Methods in Genome Research (S. Suhai,ed.), pp. 1-14, Plenum, New York (1997).

This present invention also provides expression vectors comprising theisolated nucleic acids of the invention, wherein the nucleic acid isoperably linked to control sequences that are recognized by a host cellwhen the host cell is transfected with the vector. Also provided arehost cells comprising an expression vector of the present invention andmethods for producing the antibody or antigen binding fragment thereofdisclosed herein comprising culturing a host cell harboring anexpression vector encoding the antibody or antigen binding fragment inculture medium, and isolating the antigen or antigen binding fragmentthereof from the host cell or culture medium.

Epitope Binding and Binding Affinity

The present invention further provides antibodies or antigen bindingfragments thereof that bind to amino acid residues of the same epitopeof canine IL-4R_(α) as the murine anti-canine IL-4R_(α) antibodiesdisclosed herein. In particular embodiments the murine anti-canineIL-4R_(α) antibodies or antigen binding fragments thereof are alsocapable of inhibiting/blocking the binding of canine IL-4 and canineIL-13 to the type-I and/or type II IL-4 receptors.

A caninized murine anti-canine IL-4R_(α) antibody can be producedrecombinantly by methods that are known in the field. Mammalian celllines available as hosts for expression of the antibodies or fragmentsdisclosed herein are well known in the art and include many immortalizedcell lines available from the American Type Culture Collection (ATCC).These include, inter alia, Chinese hamster ovary (CHO) cells, NSO, SP2cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells(COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells,3T3 cells, HEK-293 cells and a number of other cell lines. Mammalianhost cells include human, mouse, rat, dog, monkey, pig, goat, bovine,horse and hamster cells. Cell lines of particular preference areselected through determining which cell lines have high expressionlevels. Other cell lines that may be used are insect cell lines, such asSf9 cells, amphibian cells, bacterial cells, plant cells and fungalcells. When recombinant expression vectors encoding the heavy chain orantigen-binding portion or fragment thereof, the light chain and/orantigen-binding fragment thereof are introduced into mammalian hostcells, the antibodies are produced by culturing the host cells for aperiod of time sufficient to allow for expression of the antibody in thehost cells or, more preferably, secretion of the antibody into theculture medium in which the host cells are grown.

Antibodies can be recovered from the culture medium using standardprotein purification methods. Further, expression of antibodies of theinvention (or other moieties therefrom) from production cell lines canbe enhanced using a number of known techniques. For example, theglutamine synthetase gene expression system (the GS system) is a commonapproach for enhancing expression under certain conditions. The GSsystem is discussed in whole or part in connection with European PatentNos. 0 216 846, 0 256 055, and 0 323 997 and European Patent ApplicationNo. 89303964.4.

In general, glycoproteins produced in a particular cell line ortransgenic animal will have a glycosylation pattern that ischaracteristic for glycoproteins produced in the cell line or transgenicanimal. Therefore, the particular glycosylation pattern of an antibodywill depend on the particular cell line or transgenic animal used toproduce the antibody. However, all antibodies encoded by the nucleicacid molecules provided herein, or comprising the amino acid sequencesprovided herein, comprise the instant invention, independent of theglycosylation pattern that the antibodies may have. Similarly, inparticular embodiments, antibodies with a glycosylation patterncomprising only non-fucosylated N-glycans may be advantageous, becausethese antibodies have been shown to typically exhibit more potentefficacy than their fucosylated counterparts both in vitro and in vivo[See for example, Shinkawa et al., J. Biol. Chem. 278: 3466-3473 (2003);U.S. Pat. Nos. 6,946,292 and 7,214,775].

The present invention further includes antibody fragments of the murineanti-canine IL-4R_(a) antibodies disclosed herein. The antibodyfragments include F(ab)₂ fragments, which may be produced by enzymaticcleavage of an IgG by, for example, pepsin. Fab fragments may beproduced by, for example, reduction of F(ab)₂ with dithiothreitol ormercaptoethylamine. A Fab fragment is a V_(L)-C_(L) chain appended to aV_(H)-C_(H1) chain by a disulfide bridge. A F(ab)₂ fragment is two Fabfragments which, in turn, are appended by two disulfide bridges. The Fabportion of an F(ab)₂ molecule includes a portion of the F_(c) regionbetween which disulfide bridges are located. An F_(V) fragment is aV_(L) or V_(H) region.

In one embodiment, the antibody or antigen binding fragment comprises aheavy chain constant region, e.g., a canine constant region, such asIgG-A, IgG-B, IgG-C and IgG-D canine heavy chain constant region or avariant thereof. In another embodiment, the antibody or antigen bindingfragment comprises a light chain constant region, e.g., a canine lightchain constant region, such as lambda or kappa canine light chain regionor variant thereof. By way of example, and not limitation, the canineheavy chain constant region can be from IgG-B and the canine light chainconstant region can be from kappa.

Antibody Engineering

Caninized murine anti-canine IL-4R_(a) antibodies of the presentinvention can be engineered to include modifications to canine frameworkand/or canine frame residues within the variable domains of a parental(i.e., canine) monoclonal antibody, e.g. to improve the properties ofthe antibody.

Experimental and Diagnostic Uses

Murine anti-canine IL-4R_(a) and/or caninized murine anti-canineIL-4R_(a) antibodies or antigen-binding fragments thereof of the presentinvention may also be useful in diagnostic assays for canine IL-4R_(a)protein, e.g., detecting its expression in conjunction with and/orrelation to atopic dermatitis.

For example, such a method comprises the following steps:

(a) coat a substrate (e.g., surface of a microtiter plate well, e.g., aplastic plate) with a murine anti-canine IL-4R_(a) antibody or anantigen-binding fragment thereof;

-   -   (b) apply a sample to be tested for the presence of canine        IL-4R_(a) to the substrate;    -   (c) wash the plate, so that unbound material in the sample is        removed;    -   (d) apply detectably labeled antibodies (e.g., enzyme-linked        antibodies) which are also specific to the IL-4R_(α) antigen;    -   (e) wash the substrate, so that the unbound, labeled antibodies        are removed;    -   (f) if the labeled antibodies are enzyme linked, apply a        chemical which is converted by the enzyme into a fluorescent        signal; and    -   (g) detect the presence of the labeled antibody.

In a further embodiment, the labeled antibody is labeled with peroxidasewhich react with ABTS [e.g.,2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)] or3,3′,5,5′-Tetramethylbenzidine to produce a color change which isdetectable. Alternatively, the labeled antibody is labeled with adetectable radioisotope (e.g., ³H) which can be detected byscintillation counter in the presence of a scintillant. Murineanti-canine IL-4R_(α) antibodies of the invention may be used in aWestern blot or immuno protein blot procedure.

Such a procedure forms part of the present invention and includes forexample:

-   -   (i) contacting a membrane or other solid substrate to be tested        for the presence of bound canine IL-4R_(α) or a fragment thereof        with a murine anti-canine IL-4R_(α) antibody or antigen-binding        fragment thereof of the present invention. Such a membrane may        take the form of a nitrocellulose or vinyl-based [e.g.,        polyvinylidene fluoride (PVDF)] membrane to which the proteins        to be tested for the presence of canine IL-4R_(α) in a        non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or        SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel        electrophoresis) gel have been transferred (e.g., following        electrophoretic separation in the gel). Before contact of        membrane with the murine anti-canine IL-4R_(α) antibody or        antigen-binding fragment thereof, the membrane is optionally        blocked, e.g., with non-fat dry milk or the like so as to bind        non-specific protein binding sites on the membrane.    -   (ii) washing the membrane one or more times to remove unbound        murine anti-canine IL-4R_(α) antibody or an antigen-binding        fragment thereof and other unbound substances; and    -   (iii) detecting the bound murine anti-canine IL-4R_(α) antibody        or antigen-binding fragment thereof.

Detection of the bound antibody or antigen-binding fragment may be bybinding the antibody or antigen-binding fragment with a secondaryantibody (an anti-immunoglobulin antibody) which is detectably labeledand, then, detecting the presence of the secondary antibody.

The murine anti-canine IL-4R_(α) antibodies and antigen-bindingfragments thereof disclosed herein may also be used forimmunohistochemistry. Such a method forms part of the present inventionand comprises, e.g., (1) contacting a cell to be tested for the presenceof canine IL-4R_(α) with a murine anti-canine IL-4R_(α) antibody orantigen-binding fragment thereof of the present invention; and (2)detecting the antibody or fragment on or in the cell. If the antibody orantigen-binding fragment itself is detectably labeled, it can bedetected directly. Alternatively, the antibody or antigen-bindingfragment may be bound by a detectably labeled secondary antibody whichis detected.

Imaging techniques include SPECT imaging (single photon emissioncomputed tomography) or PET imaging (positron emission tomography).Labels include e.g., iodine-123 (¹²³1) and technetium-99m (^(99m)Tc),e.g., in conjunction with SPECT imaging or ¹¹C, ¹³N, ¹⁵O or ¹⁸F, e.g.,in conjunction with PET imaging or Indium-111 [See e.g., Gordon et al.,International Rev. Neurobiol. 67:385-440 (2005)].

Cross-Blocking Antibodies

Furthermore, an anti-canine IL-4R_(a) antibody or antigen-bindingfragment thereof of the present invention includes any antibody orantigen-binding fragment thereof that binds to the same epitope incanine IL-4R_(a) to which the antibodies and fragments discussed hereinbind and any antibody or antigen-binding fragment that cross-blocks(partially or fully) or is cross-blocked (partially or fully) by anantibody or fragment discussed herein for canine IL-4R_(a) binding; aswell as any variant thereof.

The cross-blocking antibodies and antigen-binding fragments thereofdiscussed herein can be identified based on their ability tocross-compete with the antibodies disclosed herein (on the basis of theCDRs as provided below in Example 5), i.e., 1A3, 1A9, 1B12, 10C12, 10F2,10E10, 10G8, and/or 11D3; or more particularly, 11B6 and/or 6C12; andeven more particularly 4D8, 4H3, 2E2, and/or 11H2, in standard bindingassays (e.g., BIACore®, ELISA, as exemplified below, or flow cytometry).For example, standard ELISA assays can be used in which a recombinantcanine IL-4R_(α) protein is immobilized on the plate, one of theantibodies is fluorescently labeled and the ability of non-labeledantibodies to compete off the binding of the labeled antibody isevaluated. Additionally or alternatively, BIAcore® analysis can be usedto assess the ability of the antibodies to cross-compete. The ability ofa test antibody to inhibit the binding of, for example, 1A3, 1A9, 1B12,10C12, 10F2, 10E10, 10G8, and/or 11D3; or more particularly, 11B6 and/or6C12; and even more particularly 4D8, 4H3, 2E2, and/or 11H2, to canineIL-4R_(α) demonstrates that the test antibody can compete with 1A3, 1A9,1B12, 10C12, 10F2, 10E10, 10G8, 11D3, 11B6, 6C12, 4D8, 4H3, 2E2, and/or11H2 for binding to canine IL-4R_(α) and thus, may, in some cases, bindto the same epitope on canine IL-4R_(α) as 1A3, 1A9, 1B12, 10C12, 10F2,10E10, 10G8, 11D3, 11B6, 6C12, 4D8, 4H3, 2E2, and/or 11H2. As statedabove, antibodies and fragments that bind to the same epitope as any ofthe anti-canine IL-4R_(α) antibodies or fragments of the presentinvention also form part of the present invention.

Pharmaceutical Compositions and Administration

To prepare pharmaceutical or sterile compositions of a caninized murineanti-canine IL-4R_(α) antibody or antigen binding fragment thereof itcan be admixed with a pharmaceutically acceptable carrier or excipient.[See, e.g., Remington's Pharmaceutical Sciences and U.S. Pharmacopeia:National Formulary, Mack Publishing Company, Easton, Pa. (1984)].

Formulations of therapeutic and diagnostic agents may be prepared bymixing with acceptable carriers, excipients, or stabilizers in the formof, e.g., lyophilized powders, slurries, aqueous solutions orsuspensions [see, e.g., Hardman, et al. (2001) Goodman and Gilman's ThePharmacological Basis of Therapeutics, McGraw-Hill, New York, N.Y.;Gennaro (2000) Remington: The Science and Practice of Pharmacy,Lippincott, Williams, and Wilkins, New York, N.Y.; Avis, et al. (eds.)(1993) Pharmaceutical Dosage Forms: Parenteral Medications, MarcelDekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms:Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weinerand Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc.,New York, N.Y.]. In one embodiment, anti-IL-4R_(α) antibodies of thepresent invention are diluted to an appropriate concentration in asodium acetate solution pH 5-6, and NaCl or sucrose is added fortonicity. Additional agents, such as polysorbate 20 or polysorbate 80,may be added to enhance stability.

Toxicity and therapeutic efficacy of the antibody compositions,administered alone or in combination with another agent, can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining the LD₅₀ (the dose lethal to50% of the population) and the ED₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index (LD₅₀/ED₅₀). In particular aspects,antibodies exhibiting high therapeutic indices are desirable. The dataobtained from these cell culture assays and animal studies can be usedin formulating a range of dosage for use in canines. The dosage of suchcompounds lies preferably within a range of circulating concentrationsthat include the ED₅₀ with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration.

The mode of administration can vary. Suitable routes of administrationinclude oral, rectal, transmucosal, intestinal, parenteral;intramuscular, subcutaneous, intradermal, intramedullary, intrathecal,direct intraventricular, intravenous, intraperitoneal, intranasal,intraocular, inhalation, insufflation, topical, cutaneous, transdermal,or intra-arterial. In particular embodiments, the murine anti-canineIL-4R_(α) antibody or antigen binding fragment thereof can beadministered by an invasive route such as by injection. In furtherembodiments of the invention, a murine anti-canine IL-4R_(α) antibody orantigen binding fragment thereof, or pharmaceutical composition thereof,is administered intravenously, subcutaneously, intramuscularly,intraarterially, or by inhalation, aerosol delivery. Administration bynon-invasive routes (e.g., orally; for example, in a pill, capsule ortablet) is also within the scope of the present invention.

Compositions can be administered with medical devices known in the art.For example, a pharmaceutical composition of the invention can beadministered by injection with a hypodermic needle, including, e.g., aprefilled syringe or autoinjector. The pharmaceutical compositionsdisclosed herein may also be administered with a needleless hypodermicinjection device; such as the devices disclosed in U.S. Pat. No.6,620,135; 6,096,002; 5,399,163; 5,383,851; 5,312,335; 5,064,413;4,941,880; 4,790,824 or 4,596,556.

The pharmaceutical compositions disclosed herein may also beadministered by infusion. Examples of well-known implants and modulesform administering pharmaceutical compositions include: U.S. Pat. No.4,487,603, which discloses an implantable micro-infusion pump fordispensing medication at a controlled rate; U.S. Pat. No. 4,447,233,which discloses a medication infusion pump for delivering medication ata precise infusion rate; U.S. Pat. No. 4,447,224, which discloses avariable flow implantable infusion apparatus for continuous drugdelivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments. Many other suchimplants, delivery systems, and modules are well known to those skilledin the art.

Alternately, one may administer a murine anti-canine or a caninizedmurine anti-canine IL-4R_(α) antibody in a local rather than systemicmanner, for example, via injection of the antibody directly into anarthritic joint or pathogen-induced lesion characterized byimmunopathology, often in a depot or sustained release formulation.Furthermore, one may administer the antibody in a targeted drug deliverysystem, for example, in a liposome coated with a tissue-specificantibody, targeting, for example, arthritic joint or pathogen-inducedlesion characterized by immunopathology. The liposomes will be targetedto and taken up selectively by the afflicted tissue.

The administration regimen depends on several factors, including theserum or tissue turnover rate of the therapeutic antibody, the level ofsymptoms, the immunogenicity of the therapeutic antibody, and theaccessibility of the target cells in the biological matrix. Preferably,the administration regimen delivers sufficient therapeutic antibody toeffect improvement in the target disease state, while simultaneouslyminimizing undesired side effects. Accordingly, the amount of biologicdelivered depends in part on the particular therapeutic antibody and theseverity of the condition being treated. Guidance in selectingappropriate doses of therapeutic antibodies is available [see, e.g.,Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, U K(1996); Kresina (ed.) Monoclonal Antibodies, Cytokines and Arthritis,Marcel Dekker, New York, N.Y. (1991); Bach (ed.) Monoclonal Antibodiesand Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York,N.Y. (1993); Baert, et al. New Engl. J. Med. 348:601-608 (2003); Milgromet al. New Engl. J. Med. 341:1966-1973 (1999); Slamon et al. New Engl.J. Med. 344:783-792 (2001); Beniaminovitz et al. New Engl. J. Med.342:613-619 (2000); Ghosh et al. New Engl. J. Med. 348:24-32 (2003);Lipsky et al. New Engl. J. Med. 343:1594-1602 (2000)].

Determination of the appropriate dose is made by the veterinarian, e.g.,using parameters or factors known or suspected in the art to affecttreatment. Generally, the dose begins with an amount somewhat less thanthe optimum dose and it is increased by small increments thereafteruntil the desired or optimum effect is achieved relative to any negativeside effects. Important diagnostic measures include those of symptomsof, e.g., the inflammation or level of inflammatory cytokines produced.

Antibodies or antigen binding fragments thereof disclosed herein may beprovided by continuous infusion, or by doses administered, e.g., daily,1-7 times per week, weekly, bi-weekly, monthly, bimonthly, quarterly,semiannually, annually etc. Doses may be provided, e.g., intravenously,subcutaneously, topically, orally, nasally, rectally, intramuscular,intracerebrally, intraspinally, or by inhalation. A total weekly dose isgenerally at least 0.05 μg/kg body weight, more generally at least 0.2μg/kg, 0.5 μg/kg, 1 μg/kg, 10 μg/kg, 100 μg/kg, 0.25 mg/kg, 1.0 mg/kg,2.0 mg/kg, 5.0 mg/ml, 10 mg/kg, 25 mg/kg, 50 mg/kg or more [see, e.g.,Yang, et al. New Engl. J. Med. 349:427-434 (2003); Herold, et al. NewEngl. J. Med. 346:1692-1698 (2002); Liu, et al. J. Neurol. Neurosurg.Psych. 67:451-456 (1999); Portielji, et al. Cancer Immunol. Immunother.52:133-144 (2003)]. Doses may also be provided to achieve apre-determined target concentration of a caninized murine anti-canineIL-4R_(α) antibody in the subject's serum, such as 0.1, 0.3, 1, 3, 10,30, 100, 300 μg/ml or more. In other embodiments, a caninized murineanti-canine IL-4R_(α) antibody of the present invention is administeredsubcutaneously or intravenously, on a weekly, biweekly, “every 4 weeks,”monthly, bimonthly, or quarterly basis at 10, 20, 50, 80, 100, 200, 500,1000 or 2500 mg/subject.

The antigenic peptides recognized by anti-canine IL-4R_(α) mAbs also maybe used as vaccines to elicit antibodies that block the binding ofcanine IL-4 and canine IL-13 to the type-I and type II IL-4 receptors.Such vaccines may be useful as therapeutic vaccines for diseases such asatopic dematitis. In order to use these antigenic peptides as vaccines,one or more of these peptides may be coupled chemically or through thetechniques of recombinant DNA technology to another carrier protein inorder to enhance the immunogenicity of these peptides and elicitpeptide-specific antibodies. Techniques for coupling peptides to carrierproteins are known to those skilled in the art. Peptide vaccines may beused to vaccinate animals by IM, S/C, oral, spray or in ovo routes.Peptide vaccines may be used as subunit proteins expressed frombacterial, viral, yeast or baculovirus virus systems. Alternatively suchpeptide vaccines may be delivered following administration of a varietyof viral or bacterial vectors that express such peptide vaccines as canbe practiced by methods known to those skilled in the art. The peptidevaccines may be administered in doses from 1-1000 μg and may optionallycontain an adjuvant and an acceptable pharmaceutical carrier.

As used herein, “inhibit” or “treat” or “treatment” includes apostponement of development of the symptoms associated with a disorderand/or a reduction in the severity of the symptoms of such disorder. Theterms further include ameliorating existing uncontrolled or unwantedsymptoms, preventing additional symptoms, and ameliorating or preventingthe underlying causes of such symptoms. Thus, the terms denote that abeneficial result has been conferred on a vertebrate subject with adisorder, disease or symptom, or with the potential to develop such adisorder, disease or symptom.

As used herein, the terms “therapeutically effective amount”,“therapeutically effective dose” and “effective amount” refer to anamount of a caninized murine anti-canine IL-4R_(α) antibody or antigenbinding fragment thereof of the present invention that, whenadministered alone or in combination with an additional therapeuticagent to a cell, tissue, or subject, is effective to cause a measurableimprovement in one or more symptoms of a disease or condition or theprogression of such disease or condition. A therapeutically effectivedose further refers to that amount of the binding compound sufficient toresult in at least partial amelioration of symptoms, e.g., treatment,healing, prevention or amelioration of the relevant medical condition,or an increase in rate of treatment, healing, prevention or ameliorationof such conditions. When applied to an individual active ingredientadministered alone, a therapeutically effective dose refers to thatingredient alone. When applied to a combination, a therapeuticallyeffective dose refers to combined amounts of the active ingredients thatresult in the therapeutic effect, whether administered in combination,serially or simultaneously. An effective amount of a therapeutic willresult in an improvement of a diagnostic measure or parameter by atleast 10%; usually by at least 20%; preferably at least about 30%; morepreferably at least 40%, and most preferably by at least 50%. Aneffective amount can also result in an improvement in a subjectivemeasure in cases where subjective measures are used to assess diseaseseverity.

Other Combination Therapies

As previously described, a caninized murine anti-canine IL-4R_(α)antibody or antigen binding fragment thereof and/or an antigenic peptideof the present invention may be coadministered with one or other moretherapeutic agents (such as an inhibitor as discussed in the nextparagraph) and/or a murine (or caninized murine) anti-canine TSLPantibody [see, U.S. Pat. No. 8,791,242]. The antibod(ies) may be linkedto the agent (as an immunocomplex) and/or can be administered separatelyfrom the agent or other antibody. In the latter case (separateadministration), the antibodies can be administered before, after orconcurrently with the agent or can be co-administered with other knowntherapies.

Kits

Further provided are kits comprising one or more components thatinclude, but are not limited to, an antibody or antigen bindingfragment, as discussed herein, which specifically binds IL-4R_(α) (e.g.,a caninized murine anti-canine IL-4R_(α) antibody or antigen bindingfragment thereof) in association with one or more additional componentsincluding, but not limited to a pharmaceutically acceptable carrierand/or an inhibitor such as a Janus kinase (JAK) inhibitor, e.g.,oclacitinib [see, WO 2013/040241], a spleen tyrosine kinase (SYK)inhibitor [see e.g., U.S. Pat. No. 8,759,366], or an antagonist to achemoattractant receptor-homologous molecule expressed on TH2 cells [seee.g., WO 2010/099039; WO 2010/031183; and U.S. Pat. No. 8,546,422]. Thebinding composition and/or an inhibitor, as described directly above,can be formulated as a pure composition or in combination with apharmaceutically acceptable carrier, in a pharmaceutical composition.

In one embodiment, the kit includes a binding composition of the presentinvention (e.g., a caninized murine anti-canine IL-4R_(α) or apharmaceutical composition thereof in one container (e.g., in a sterileglass or plastic vial) and a pharmaceutical composition thereof and/oran inhibitor as described above in another container (e.g., in a sterileglass or plastic vial).

If the kit includes a pharmaceutical composition for parenteraladministration to a subject, the kit can also include a device forperforming such administration. For example, the kit can include one ormore hypodermic needles or other injection devices as discussed above.The kit can also include a package insert including informationconcerning the pharmaceutical compositions and dosage forms in the kit.Generally, such information aids pet owners and veterinarians in usingthe enclosed pharmaceutical compositions and dosage forms effectivelyand safely. For example, the following information regarding acombination of the invention may be supplied in the insert:pharmacokinetics, pharmacodynamics, clinical studies, efficacyparameters, indications and usage, contraindications, warnings,precautions, adverse reactions, overdosage, proper dosage andadministration, how supplied, proper storage conditions, references,manufacturer/distributor information and patent information.

As a matter of convenience, an antibody or specific binding agentdisclosed herein can be provided in a kit, i.e., a packaged combinationof reagents in predetermined amounts with instructions for performingthe diagnostic or detection assay. Where the antibody is labeled with anenzyme, the kit will include substrates and cofactors required by theenzyme (e.g., a substrate precursor which provides the detectablechromophore or fluorophore). In addition, other additives may beincluded such as stabilizers, buffers (e.g., a block buffer or lysisbuffer) and the like. The relative amounts of the various reagents maybe varied widely to provide for concentrations in solution of thereagents which substantially optimize the sensitivity of the assay.Particularly, the reagents may be provided as dry powders, usuallylyophilized, including excipients which on dissolution will provide areagent solution having the appropriate concentration.

EXAMPLES Example 1 Identification and Cloning of Canine IL-4 Receptor αChain Receptor

The cDNA encoding a predicted full length canine IL-4 receptor alphachain (SEQ ID NO: 1) was identified through a search of the Genbankdatabase (accession #XM_547077.4; see also, U.S. Pat. No. 7,208,579 B2).This predicted cDNA encodes an 823 amino acids (SEQ ID NO: 2) includinga 25 amino acid leader sequence and is identified as accession#XP_547077.3. The mature predicted canine IL-4 receptor α chain protein(SEQ ID NO: 4) shares 65% identity with human IL-4 receptor α chain(accession #NP_000409.1) and 70% identity with swine IL-4 receptor αchain (accession #NP_999505.1). The mature predicted canine IL-4receptor α chain protein is encoded by the nucleotide sequenceidentified as SEQ ID NO: 3. Comparison of the predicted mature IL-4receptor α chain with the known sequences of human IL-4 receptor α chainidentified the extracellular domain (ECD) of the mature canine IL-4receptor α chain protein and is designated as SEQ ID NO: 6. The DNAsequence encoding the ECD of the mature canine IL-4 receptor α chain isidentified as SEQ ID NO: 5.

Canine IL-4 receptor α chain full length DNA with signal sequence(SEQ ID NO: 1):atgggcagactgtgcagcggcctgaccttccccgtgagctgcctggtgctggtgtgggtggccagcagcggcagcgtgaaggtgctgcacgagcccagctgcttcagcgactacatcagcaccagcgtgtgccagtggaagatggaccaccccaccaactgcagcgccgagctgagactgagctaccagctggacttcatgggcagcgagaaccacacctgcgtgcccgagaacagagaggacagcgtgtgcgtgtgcagcatgcccatcgacgacgccgtggaggccgacgtgtaccagctggacctgtgggccggccagcagctgctgtggagcggcagcttccagcccagcaagcacgtgaagcccagaacccccggcaacctgaccgtgcaccccaacatcagccacacctggctgctgatgtggaccaacccctaccccaccgagaaccacctgcacagcgagctgacctacatggtgaacgtgagcaacgacaacgaccccgaggacttcaaggtgtacaacgtgacctacatgggccccaccctgagactggccgccagcaccctgaagagcggcgccagctacagcgccagagtgagagcctgggcccagacctacaacagcacctggagcgactggagccccagcaccacctggctgaactactacgagccctgggagcagcacctgcccctgggcgtgagcatcagctgcctggtgatcctggccatctgcctgagctgctacttcagcatcatcaagatcaagaagggctggtgggaccagatccccaaccccgcccacagccccctggtggccatcgtgatccaggacagccaggtgagcctgtggggcaagagaagcagaggccaggagcccgccaagtgcccccactggaagacctgcctgaccaagctgctgccctgcctgctggagcacggcctgggcagagaggaggagagccccaagaccgccaagaacggccccctgcagggccccggcaagcccgcctggtgccccgtggaggtgagcaagaccatcctgtggcccgagagcatcagcgtggtgcagtgcgtggagctgagcgaggcccccgtggacaacgaggaggaggaggaggtggaggaggacaagagaagcctgtgccccagcctggagggcagcggcggcagcttccaggagggcagagagggcatcgtggccagactgaccgagagcctgttcctggacctgctgggcggcgagaacggcggcttctgcccccagggcctggaggagagctgcctgcccccccccagcggcagcgtgggcgcccagatgccctgggcccagttccccagagccggccccagagccgcccccgagggccccgagcagcccagaagacccgagagcgccctgcaggccagccccacccagagcgccggcagcagcgccttccccgagcccccccccgtggtgaccgacaaccccgcctacagaagcttcggcagcttcctgggccagagcagcgaccccggcgacggcgacagcgaccccgagctggccgacagacccggcgaggccgaccccggcatccccagcgccccccagccccccgagccccccgccgccctgcagcccgagcccgagagctgggagcagatcctgagacagagcgtgctgcagcacagagccgcccccgcccccggccccggccccggcagcggctacagagagttcacctgcgccgtgaagcagggcagcgcccccgacgccggcggccccggcttcggccccagcggcgaggccggctacaaggccttctgcagcctgctgcccggcggcgccacctgccccggcaccagcggcggcgaggccggcagcggcgagggcggctacaagcccttccagagcctgacccccggctgccccggcgcccccacccccgtgcccgtgcccctgttcaccttcggcctggacaccgagccccccggcagcccccaggacagcctgggcgccggcagcagccccgagcacctgggcgtggagcccgccggcaaggaggaggacagcagaaagaccctgctggcccccgagcaggccaccgaccccctgagagacgacctggccagcagcatcgtgtacagcgccctgacctgccacctgtgcggccacctgaagcagtggcacgaccaggaggagagaggcaaggcccacatcgtgcccagcccctgctgcggctgctgctgcggcgacagaagcagcctgctgctgagccccctgagagcccccaacgtgctgcccggcggcgtgctgctggaggccagcctgagccccgccagcctggtgcccagcggcgtgagcaaggagggcaagagcagccccttcagccagcccgccagcagcagcgcccagagcagcagccagacccccaagaagctggccgtgctgagcaccgagcccacctgcatgagcgccagcCanine IL-4 receptor α full length protein with signal sequence in bold font(SEQ ID NO: 2).MGRLCSGLTFPVSCLVLVWVASSGSVKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDDAVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVNVSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNYYEPWEQHLPLGVSISCLVILAICLSCYFSIIKIKKGWWDQIPNPAHSPLVAIVIQDSQVSLWGKRSRGQEPAKCPHWKTCLTKLLPCLLEHGLGREEESPKTAKNGPLQGPGKPAWCPVEVSKTILWPESISVVQCVELSEAPVDNEEEEEVEEDKRSLCPSLEGSGGSFQEGREGIVARLTESLELDLLGGENGGFCPQGLEESCLPPPSGSVGAQMPWAQFPRAGPRAAPEGPEQPRRPESALQASPTQSAGSSAFPEPPPVVTDNPAYRSEGSFLGQSSDPGDGDSDPELADRPGEADPGIPSAPQPPEPPAALQPEPESWEQILRQSVLQHRAAPAPGPGPGSGYREFTCAVKQGSAPDAGGPGFGPSGEAGYKAFCSLLPGGATCPGTSGGEAGSGEGGYKPFQSLTPGCPGAPTPVPVPLFTFGLDTEPPGSPQDSLGAGSSPEHLGVEPAGKEEDSRKTLLAPEQATDPLRDDLASSIVYSALTCHLCGHLKQWHDQEERGKAHIVPSPCCGCCCGDRSSLLLSPLRAPNVLPGGVLLEASLSPASLVPSGVSKEGKSSPFSQPASSSAQSSSQTPKKLAVLSTEPTCMSASCanine IL-4 receptor mature full length protein without signal sequence(SEQ ID NO: 4)VKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDDAVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVNVSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNYYEPWEQHLPLGVSISCLVILAICLSCYFSIIKIKKGWWDQIPNPAHSPLVAIVIQDSQVSLWGKRSRGQEPAKCPHWKTCLTKLLPCLLEHGLGREEESPKTAKNGPLQGPGKPAWCPVEVSKTILWPESISVVQCVELSEAPVDNEEEEEVEEDKRSLCPSLEGSGGSFQEGREGIVARLTESLFLDLLGGENGGFCPQGLEESCLPPPSGSVGAQMPWAQFPRAGPRAAPEGPEQPRRPESALQASPTQSAGSSAFPEPPPVVTDNPAYRSFGSFLGQSSDPGDGDSDPELADRPGEADPGIPSAPQPPEPPAALQPEPESWEQILRQSVLQHRAAPAPGPGPGSGYREFTCAVKQGSAPDAGGPGFGPSGEAGYKAFCSLLPGGATCPGTSGGEAGSGEGGYKPFQSLTPGCPGAPTPVPVPLFTFGLDTEPPGSPQDSLGAGSSPEHLGVEPAGKEEDSRKTLLAPEQATDPLRDDLASSIVYSALTCHLCGHLKQWHDQEERGKAHIVPSPCCGCCCGDRSSLLLSPLRAPNVLPGGVLLEASLSPASLVPSGVSKEGKSSPFSQPASSSAQSSSQTPKKLAVLSTEPTCMSASCanine IL-4 receptor mature full length DNA without signal sequence(SEQ ID NO: 3)gtgaaggtgctgcacgagcccagctgcttcagcgactacatcagcaccagcgtgtgccagtggaagatggaccaccccaccaactgcagcgccgagctgagactgagctaccagctggacttcatgggcagcgagaaccacacctgcgtgcccgagaacagagaggacagcgtgtgcgtgtgcagcatgcccatcgacgacgccgtggaggccgacgtgtaccagctggacctgtgggccggccagcagctgctgtggagcggcagcttccagcccagcaagcacgtgaagcccagaacccccggcaacctgaccgtgcaccccaacatcagccacacctggctgctgatgtggaccaacccctaccccaccgagaaccacctgcacagcgagctgacctacatggtgaacgtgagcaacgacaacgaccccgaggacttcaaggtgtacaacgtgacctacatgggccccaccctgagactggccgccagcaccctgaagagcggcgccagctacagcgccagagtgagagcctgggcccagacctacaacagcacctggagcgactggagccccagcaccacctggctgaactactacgagccctgggagcagcacctgcccctgggcgtgagcatcagctgcctggtgatcctggccatctgcctgagctgctacttcagcatcatcaagatcaagaagggctggtgggaccagatccccaaccccgcccacagccccctggtggccatcgtgatccaggacagccaggtgagcctgtggggcaagagaagcagaggccaggagcccgccaagtgcccccactggaagacctgcctgaccaagctgctgccctgcctgctggagcacggcctgggcagagaggaggagagccccaagaccgccaagaacggccccctgcagggccccggcaagcccgcctggtgccccgtggaggtgagcaagaccatcctgtggcccgagagcatcagcgtggtgcagtgcgtggagctgagcgaggcccccgtggacaacgaggaggaggaggaggtggaggaggacaagagaagcctgtgccccagcctggagggcagcggcggcagcttccaggagggcagagagggcatcgtggccagactgaccgagagcctgttcctggacctgctgggcggcgagaacggcggcttctgcccccagggcctggaggagagctgcctgcccccccccagcggcagcgtgggcgcccagatgccctgggcccagttccccagagccggccccagagccgcccccgagggccccgagcagcccagaagacccgagagcgccctgcaggccagccccacccagagcgccggcagcagcgccttccccgagcccccccccgtggtgaccgacaaccccgcctacagaagcttcggcagcttcctgggccagagcagcgaccccggcgacggcgacagcgaccccgagctggccgacagacccggcgaggccgaccccggcatccccagcgccccccagccccccgagccccccgccgccctgcagcccgagcccgagagctgggagcagatcctgagacagagcgtgctgcagcacagagccgcccccgcccccggccccggccccggcagcggctacagagagttcacctgcgccgtgaagcagggcagcgcccccgacgccggcggccccggcttcggccccagcggcgaggccggctacaaggccttctgcagcctgctgcccggcggcgccacctgccccggcaccagcggcggcgaggccggcagcggcgagggcggctacaagcccttccagagcctgacccccggctgccccggcgcccccacccccgtgcccgtgcccctgttcaccttcggcctggacaccgagccccccggcagcccccaggacagcctgggcgccggcagcagccccgagcacctgggcgtggagcccgccggcaaggaggaggacagcagaaagaccctgctggcccccgagcaggccaccgaccccctgagagacgacctggccagcagcatcgtgtacagcgccctgacctgccacctgtgcggccacctgaagcagtggcacgaccaggaggagagaggcaaggcccacatcgtgcccagcccctgctgcggctgctgctgcggcgacagaagcagcctgctgctgagccccctgagagcccccaacgtgctgcccggcggcgtgctgctggaggccagcctgagccccgccagcctggtgcccagcggcgtgagcaaggagggcaagagcagccccttcagccagcccgccagcagcagcgcccagagcagcagccagacccccaagaagctggccgtgctgagcaccgagcccacctgcatgagcgccagcCanine IL-4 receptor α chain extracellular protein domain without the signalsequence (SEQ ID NO: 6):VKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDDAVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVNVSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNYYEPWEQHLPCanine IL-4 receptor α chain extracellular DNA domain without the signal sequence (SEQ ID NO: 5):gtgaaggtgctgcacgagcccagctgcttcagcgactacatcagcaccagcgtgtgccagtggaagatggaccaccccaccaactgcagcgccgagctgagactgagctaccagctggacttcatgggcagcgagaaccacacctgcgtgcccgagaacagagaggacagcgtgtgcgtgtgcagcatgcccatcgacgacgccgtggaggccgacgtgtaccagctggacctgtgggccggccagcagctgctgtggagcggcagcttccagcccagcaagcacgtgaagcccagaacccccggcaacctgaccgtgcaccccaacatcagccacacctggctgctgatgtggaccaacccctaccccaccgagaaccacctgcacagcgagctgacctacatggtgaacgtgagcaacgacaacgaccccgaggacttcaaggtgtacaacgtgacctacatgggccccaccctgagactggccgccagcaccctgaagagcggcgccagctacagcgccagagtgagagcctgggcccagacctacaacagcacctggagcgactggagccccagcaccacctggctgaactactacgagccctgggagcagcacctgcccCanine IL-4 receptor α chain extracellular domain with a c-terminal 8 HIS Tag(SEQ ID NO: 8):VKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDDAVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVNVSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNYYEPWEQHLPHHHHHHHHCanine IL-4 receptor α chain extracellular DNA domain with a c-terminal 8 HISTag (SEQ ID NO: 7):gtgaaggtgctgcacgagcccagctgcttcagcgactacatcagcaccagcgtgtgccagtggaagatggaccaccccaccaactgcagcgccgagctgagactgagctaccagctggacttcatgggcagcgagaaccacacctgcgtgcccgagaacagagaggacagcgtgtgcgtgtgcagcatgcccatcgacgacgccgtggaggccgacgtgtaccagctggacctgtgggccggccagcagctgctgtggagcggcagcttccagcccagcaagcacgtgaagcccagaacccccggcaacctgaccgtgcaccccaacatcagccacacctggctgctgatgtggaccaacccctaccccaccgagaaccacctgcacagcgagctgacctacatggtgaacgtgagcaacgacaacgaccccgaggacttcaaggtgtacaacgtgacctacatgggccccaccctgagactggccgccagcaccctgaagagcggcgccagctacagcgccagagtgagagcctgggcccagacctacaacagcacctggagcgactggagccccagcaccacctggctgaactactacgagccctgggagcagcacctgccccaccaccaccaccaccaccaccacCanine IL-4 receptor α chain extracellular domain plus human IgG1 Fc(SEQ ID NO: 10):VKVLHEPSCFSDYISTSVCQWKMDHPTNCSAELRLSYQLDFMGSENHTCVPENREDSVCVCSMPIDDAVEADVYQLDLWAGQQLLWSGSFQPSKHVKPRTPGNLTVHPNISHTWLLMWTNPYPTENHLHSELTYMVNVSNDNDPEDFKVYNVTYMGPTLRLAASTLKSGASYSARVRAWAQTYNSTWSDWSPSTTWLNYYEPWEQHLEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKCanine IL-4 receptor α chain extracellular DNA domain plus human IgG1 Fc(SEQ ID NO: 9):gtgaaggtgctgcacgagcccagctgcttcagcgactacatcagcaccagcgtgtgccagtggaagatggaccaccccaccaactgcagcgccgagctgagactgagctaccagctggacttcatgggcagcgagaaccacacctgcgtgcccgagaacagagaggacagcgtgtgcgtgtgcagcatgcccatcgacgacgccgtggaggccgacgtgtaccagctggacctgtgggccggccagcagctgctgtggagcggcagcttccagcccagcaagcacgtgaagcccagaacccccggcaacctgaccgtgcaccccaacatcagccacacctggctgctgatgtggaccaacccctaccccaccgagaaccacctgcacagcgagctgacctacatggtgaacgtgagcaacgacaacgaccccgaggacttcaaggtgtacaacgtgacctacatgggccccaccctgagactggccgccagcaccctgaagagcggcgccagctacagcgccagagtgagagcctgggcccagacctacaacagcacctggagcgactggagccccagcaccacctggctgaactactacgagccctgggagcagcacctggagcccaagagctgcgacaagacccacacctgccccccctgccccgcccccgagctgctgggcggccccagcgtgttcctgttcccccccaagcccaaggacaccctgatgatcagcagaacccccgaggtgacctgcgtggtggtggacgtgagccacgaggaccccgaggtgaagttcaactggtacgtggacggcgtggaggtgcacaacgccaagaccaagcccagagaggagcagtacaacagcacctacagagtggtgagcgtgctgaccgtgctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtgagcaacaaggccctgcccgcccccatcgagaagaccatcagcaaggccaagggccagcccagagagccccaggtgtacaccctgccccccagcagagacgagctgaccaagaaccaggtgagcctgacctgcctggtgaagggcttctaccccagcgacatcgccgtggagtgggagagcaacggccagcccgagaacaactacaagaccaccccccccgtgctggacagcgacggcagcttcttcctgtacagcaagctgaccgtggacaagagcagatggcagcagggcaacgtgttcagctgcagcgtgatgcacgaggccctgcacaaccactacacccagaagagcctgagcctgagccccggcaag

Example 2 Murine Anti-Canine IL-4 Receptor Alpha Chain Antibodies

Generation of Anti-Canine 11-4 Receptor α Chain Monoclonal Antibodies:

A total of three Balb/c mice were immunized multiple times (with 10 μgeach time) over a 17 day period. The immunizing antigen was the canineIL-4 R alpha chain extracellular domain (ECD)-human Fc fusion protein.Following immunization, serum was collected from each mouse and testedfor reactivity with canine IL-4 receptor alpha chain ECD HIS-taggedprotein. The spleen cells of the mouse with the highest serum anti-IL-4receptor alpha chain ECD titer were fused to the myeloma P3X63Ag8.653cell line. Approximately 2 weeks following fusion, supernatant fromputative hybridoma cells were tested by ELISA for their reactivity tothe IL-4 receptor alpha chain ECD HIS-tagged protein. Hybridomasproducing strong positive signals in the ELISA were subcloned bylimiting dilution and tested again for reactivity to canine IL-4receptor alpha chain ECD HIS-tagged protein.

Confirmation of Monoclonal Antibodies Reactivity Against Canine IL-4Receptor a Chain:

The reactivity of antibodies secreted by hybridomas to ECD of canineIL-4 receptor alpha chain was confirmed by ELISA. Hybridoma cells werecultured using CELLine bioreactors (Integra-biosciences) for 10-30 days.Cells were initially maintained in DMEM supplemented with 4 mML-glutamine and 10% Ultra Low IgG fetal bovine serum (FBS) from Gibco.Hybridoma cells were seeded in CELLine bioreactor cell chambers at acell density of approximately 2×10⁶ cells/mL in 15 mL of the same mediumwith the FBS concentration increased to 20%. The outer chamber wasfilled with 1 L of nutrient medium (DMEM with 4 mM L-glutamine and 2%standard FBS). Hybridoma cells in the cell chamber were expanded toapproximately 2.5×10⁷ cells/mL over 3-7 days. Then, 10 mL of cellsuspension was harvested from the cell chamber and replaced with freshmedia to allow for re-expansion of cells and subsequent harvests. Thisprocedure was repeated as necessary to obtain adequate amounts of mAbfrom each hybridoma clone. Harvested cell suspensions were centrifugedand the supernatants were filtered through 0.2 micron filter membranes.For antibody purification, each clone's supernatant was purified using aProtein G Sepharose 4 Fast flow 5 mL column (GE Healthcare) by gravityflow. After washing with Tris-EDTA (TE) buffer pH 8.0, bound antibodieswere eluted using 0.1 M glycine buffer, pH 2.7, followed by pHneutralization using 1 M Tris, pH 8.0. Antibodies were concentrated andbuffer exchanged into phosphate-buffered saline (PBS) using CentriprepYM-10 kDa NMWL centrifugal filter units (Millipore). Antibodyconcentrations were quantified using spectrophotometry. Purifiedanti-canine IL-4 receptor α chain mAbs were tested for reactivity withthe HIS-tagged ECD domain of canine IL-4 receptor alpha chain by ELISAas follows: HIS-tagged canine IL-4 receptor alpha chain protein isdiluted to 10 μg/mL in coating buffer (Carbonate/Bicarbonate pH 9.0) anddispensed at 100 μL/well in 96-well flat bottomed ELISA plates (NUNC).The plates are incubated at 4° C. overnight. The plates are then washedthree times with phosphate buffered saline containing 0.05% Tween-20(PBST). Next, 200 μl of blocking buffer (5% skim milk in PBST) is addedto each well and the plates are incubated at 37° C. for 60 minutes. Theplates are then washed three times with PBST. Next, 100 μl of test mAbsdiluted in blocking buffer is added to the first wells of theappropriate columns. Test mAbs are then diluted three-fold to theappropriate plate position. Following incubation of the plates at 37° C.for 60 minutes, the plates are washed three times with PBST. Next, 100μl per well of a 1:2,000 dilution of a horseradish peroxidase conjugatedgoat anti-mouse IgG (KPL) is added to the plates, which are thenincubated at 37° C. for 60 minutes. Then the plates are washed threetimes with PBST, and 100 μL/well of 3,3′,5,5′ tetramethyl benzidine,(TMB) substrate (from KPL) is added to the plates. The color reaction isallowed to develop for 5-20 minutes at 37° C. prior to measuringabsorbance at 650 nm.

Various mouse anti-canine IL-4R_(α) monoclonal antibodies (mAbs) wereassayed by ELISA for their ability to bind the extracellular domain ofcanine IL-4R_(α). As depicted in FIG. 1, a majority of these mAbsexhibit positive dosage-dependent binding.

Example 3 Identification of the DNA and Predicted Protein Sequences ofthe Heavy and Light Chains Variable Domains of Anti-Canine IL-4 ReceptorAlpha Chain Monoclonal Antibodies

The DNA sequence of mouse VH and VL chains are identified followingisolation of mRNA from each hybridoma using standard molecular biologymethods. The SEQ ID NOs. of the DNA and predicted amino acid sequencesof the VH and VL from these hybridomas are listed below. The DNAencoding the signal sequence and the amino acids corresponding topredicted signal sequence are underlined, those corresponding to theCDRs are in bold, and the FRs are neither underlined nor in bold (i.e.,signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4).

mAb 1A3 Heavy chain: DNA sequence (SEQ ID NO: 11):ATGGACTCCAGGCTCAATTTAGTTTTCCTTGTCCTTATTTTAAAAGGTGTCCGGTGTGAGGTGCAGCTGGTGGAGTCTGGGGGAGACTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTTTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGGGTGGGTTGCATACATTAGTAGTGGCAGTGGTACCATCTACTATGCAGACACAGTGAGGGGCCGATTCACCATCTCCAGAGACAATGTCAAGAACACCCTGTTCCTGCAAATGACCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGTAAGGGGGGACCTTTACTACGGTAGTAGTTTCGATGCTTATTGGGGCCGAGGGACTCTGGTCACTGTCTCTGCAHeavy chain: Amino acid sequence (SEQ ID NO: 12):MDSRLNLVFLVLILKGVRCEVQLVESGGDLVKPGGSLKLSCAASGFTFSDFGMHWVRQAPEKGLGWVAYISSGSGTIYYADTVRGRFTISRDNVKNTLFLQMTSLRSEDTAMYYCVRGDLYYGSSFDAYWGRGTLVTVSALight chain: DNA sequence (SEQ ID NO: 13):ATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCTTCAGTCATAATGTCCAGAGGACAAATTGTTCTCTCCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACAATGACTTGCAGGGCCAGCTCAAGTGTAAGTTTCATGTTCTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATGACACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGAGTGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTAACCCACTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAALight chain: Amino acid sequence (SEQ ID NO: 14):MDFQVQIFSFLLISASVIMSRGQIVLSQSPAILSASPGEKVTMTCRASSSVSFMFWYQQKPGSSPKPWIYDTSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSSNPLTFGAGTKLELK mAb 1A9Heavy chain: DNA sequence (SEQ ID NO: 15):ATGGAATGGCCTTGTATCTTTCTCTTCCTCCTGTCAGTAACTGAAGGTGTCCACTCCCAGGTTCCGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCCTCAGTGAAGATTTCCTGCAAGGCTTCTGGCTACGCATTCAGTAGCTCCTGGATGAACTGGGTGAAGCAGAGGCCTGGAAAGGGTCTTGAGTGGATTGGACGGATTTATCCTGGAGATGGAGATACTAAGTACAATGGGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCCTGACATCGGAGGACTCTGCGGTTTACTTCTGTGCAAGAGATGATTACGACGAGGCTTCCTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA Heavy chain: Amino acid sequence (SEQ ID NO: 16):MEWPCIFLFLLSVTEGVHSQVPLQQSGPELVKPGASVKISCKASGYAFSSSWMNWVKQRPGKGLEWIGRIYPGDGDTKYNGKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARDDYDEASWGQGTLVTVSALight chain: DNA sequence (SEQ ID NO: 17):ATGGGCATCAAGATGGAGTTTCAGACCCAGGTCTTTGTATTCGTGTTGCTCTGGTTGTCTGGTGTTGATGGAGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGAATGTTCGTTCTGCTGTAGCCTGGTATCAACAGAAACCAGGGCAGTCTCCTAAATCACTGATTTACTTGGCATCCAACCGGCACACTGGAGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATTAGCAATGTGCAATCTGAAGACCTGGCAGATTATTTCTGTCTGCAACATTGGAATTATCCATTCACGTTCGGCTCGGGGACAAAGTTGGAAATAAAA Light chain: Amino acid sequence (SEQ ID NO: 18):MGIKMEFQTQVFVFVLLWLSGVDGDIVMTQSQKFMSTSVGDRVSITCKASQNVRSAVAWYQQKPGQSPKSLIYLASNRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFCLQHWNYPFTFGSGTKLEIK mAb 1B12Heavy chain: DNA sequence (SEQ ID NO: 19):ATGGGATGGAGCTGGATCTTTCTCTTTCTCCTGTCAGGAACTGCAGGTGTCCTCTCTGAGGTCCAGCTGCAACAATCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGATATCCTGTAAGGCTTCTGGATACACGTTCACTGACTATTACATGAACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGAGACATTATTCCTAGCAATGGTGGTACTAGCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAGTCCTCCAGCGCAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCAGTCTATTACTGTGCAAGAGGGATCAGCTACTATGGTAACCGATATTACTTTACTATGGACTATTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAHeavy chain: Amino acid sequence (SEQ ID NO: 20):MGWSWIFLFLLSGTAGVLSEVQLQQSGPELVKPGASVKISCKASGYTFTDYYMNWVKQSHGKSLEWIGDIIPSNGGTSYNQKFKGKATLTVDKSSSAAYMELRSLTSEDSAVYYCARGISYYGNRYYFTMDYWGQGTSVTVSSLight chain: DNA sequence (SEQ ID NO: 21):ATGAGGTGCCTAGCTGAGTTCCTGGGGCTGCTTGTGCTCTGGATCCCTGGAGCCATTGGGGATATTGTGATGACTCAGGCTGCACCCTCTGTACCTGTCACTCCTGGAGAGTCAGTATCCATCTCCTGCAGGTCTAGTAAGAGTCTCCTGCATAGTAATGGCAACACTTACTTGTTTTGGTTCGTGCAGAGGCCAGGCCAGTCTCCTCAGCTCCTGATATATCGGATGTCCAACCTTGCCTCAGGAGTCCCAGACAGGTTCAGTGGCAGTGGGTCAGGAACTGCTTTCACACTGAGAATCAGTAGAGTGGAGGCTGAGGATGTGGGTGTTTATTACTGTATGCAACATCTAGAATATCCATTCACGTTCGGCTCGGGGACAAAGTTGGACATAAAA Light chain: Amino acid sequence (SEQ ID NO: 22):MRCLAEFLGLLVLWIPGAIGDIVMTQAAPSVPVTPGESVSISCRSSKSLLHSNGNTYLFWFVQRPGQSPQLLIYRMSNLASGVPDRFSGSGSGTAFTLRISRVEAEDVGVYYCMQHLEYPFTFGSGTKLDIK mAb 10C12Heavy chain: DNA sequence (SEQ ID NO: 23):ATGGAATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGTAACTGCAGGTGTCCAATCCCAGGTTCAACTGCAGCAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGTGTGAAGCAGACACCTGTGCACGGCCTGGAATGGATTGGAGCTATTGATCCTGAAACTTGTGGTACTGCCTACAATCAGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGATCGAAACTGGGACGAGGGTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCAHeavy chain: Amino acid sequence (SEQ ID NO: 24):MEWSWIFLFLLSVTAGVQSQVQLQQSGAELVRPGASVKLSCKASGYTFTDYEMHCVKQTPVHGLEWIGAIDPETCGTAYNQKFKGKATLTADKSSSTAYMELRSLTSEDSAVYYCTRSKLGRGWYFDVWGTGTTVTVSSLight chain: DNA sequence (SEQ ID NO: 25):ATGGAATCACAGACCCAGGTCCTCATGTTTCTTCTGCTCTGGGTATCTGGTGCCTGTGCAGACATTGTGATGACACAGTCTCCATCCTCCCTGGCTATGTCAGTAGGACAGAAGGTCACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTAAATAGTAGCAATCAAAAGAACTATTTGGCCTGGTACCAGCAGAAACCAGGACAGTCTCCTAAACTTCTGGTATACTTTGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCATAGGCAGTGGATCTGGGACAGATTTCACTCTTACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGATTACTTCTGTCAGCAACATTATAGCACTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA Light chain: Amino acid sequence (SEQ ID NO: 26):MESQTQVLMFLLLWVSGACADIVMTQSPSSLAMSVGQKVTMSCKSSQSLLNSSNQKNYLAWYQQKPGQSPKLLVYFASTRESGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQHYSTPYTFGGGTKLEIK mAb 10F2Heavy chain: DNA sequence (SEQ ID NO: 27):ATGGCTGTCCTGGCACTGCTCCTCTGCCTGGTGACATTCCCAAACTGTGTCCTGTCCCAGGTGCACCTGAAGGAGTCAGGACCTGGCCTGGTGGCGCCCTCACAGAGCCTGTCCATCACATGCACTGTCTCAGGGTTCTCTTTAACCAGCTATGGTGTAAGCTGGGTTCGCCAGCCTCCAGGAGAGGGTCTGGAGTGGCTGGGAGTAATATGGGGTGACGGGAGCACATATTTTCATTCAGCTCTCATATCCAGACTGAGCATCAGCAAGGATGACTCCAAGAGCCAAGTTTTCTTAAAATTGAACAGTCTACAAACTGATGACACAGCCACGTACTACTGTGCCAAACAAGGGACGATCTATGATGGTTACTACAACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAHeavy chain: Amino acid sequence (SEQ ID NO: 28):MAVLALLLCLVTFPNCVLSQVHLKESGPGLVAPSQSLSITCTVSGFSLTSYGVSWVRQPPGEGLEWLGVIWGDGSTYFHSALISRLSISKDDSKSQVFLKLNSLQTDDTATYYCAKQGTIYDGYYNYAMDYWGQGTSVTVSSLight chain: DNA sequence (SEQ ID NO: 29):ATGGATTCACAGGCCCAGGTTCTTATGTTACTGCTGCTATGGGTATCTGGTACCTGTGGGGACATTGTGATGTCACAGTCTCCATCCTCCCTAACTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAACCTTTTATATGGTGGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAGGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATGACTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA Light chain: Amino acid sequence (SEQ ID NO: 30):MDSQAQVLMLLLLWVSGTCGDIVMSQSPSSLTVSVGEKVTMSCKSSQNLLYGGNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVRAEDLAVYYCQQYYDYPYTFGGGTKLEIK mAb 10E10Heavy chain: DNA sequence (SEQ ID NO: 31):ATGGGATGGAGCTGGATCTTTCTCTTCCTCCTGTCAGGAACTGCAGGTGTCCACTCCCAGGTTCAGCTGCAGCAGTCTGGACCTGAGCTGGTGAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTCACAACCTACGATATACACTGGGTGAAGCAGAGGCCTGGGCAGGGCCTTGAGTGGATTGGATGGATTTATCCTAGAGATGGTCGTACTACTTACAATGAGAAGTTCAAGGCCAAGGCCACATTGACTGTAGACACATCCTCCACCACAGCGTACATGGAGCTCCACAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCGAGAAGTAGCCCCTTTGGCTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA Heavy chain: Amino acid sequence (SEQ ID NO: 32):MGWSWIFLFLLSGTAGVHSQVQLQQSGPELVKPGASVKLSCKASGYTFTTYDIHWVKQRPGQGLEWIGWIYPRDGRTTYNEKFKAKATLTVDTSSTTAYMELHSLTSEDSAVYFCARSSPFGYWGQGTTLTVSSLight chain: DNA sequence (SEQ ID NO: 33):ATGAAGTTTCCTTCTCAACTTCTGCTCTTCCTGCTGTTCAGAATCACAGGCATAATATGTGACATCCAGATGACACAATCTTCATCCTACTTGTCTGTATCTCTAGGAGGCAGAGTCACCATTACTTGCAAGGCAAGTGACCACATTAATAATTGGTTAGCCTGGTATCAGCAGAAACCAGGAAATGCTCCTAGGCTCTTAATATCTGGTGCAACCAGTTTGGAAACTGGGGTTCCTTCAAGATTCAGTGGCAGTGGATCTGGAAAGGATTACACTCTCAGCATTACCAGTCTTCAGACTGAAGATGCTGCTACTTATCACTGTCACCAGTATTGGAGTATTCCGTACACGTTCGGAGGGGGGACCAAGGTGGAAATAAAALight chain: Amino acid sequence (SEQ ID NO: 34):MKFPSQLLLFLLFRITGIICDIQMTQSSSYLSVSLGGRVTITCKASDHINNWLAWYQQKPGNAPRLLISGATSLETGVPSRFSGSGSGKDYTLSITSLQTEDAATYHCHQYWSIPYTFGGGTKVEIK mAb 10G8Heavy chain: DNA sequence (SEQ ID NO: 35):ATGGAATGGAGCTGGGTCTTTCTCTTCCTCCTGTCAGTAATTGCAGGTGTCCAATCCCAGGTTCAACTGCAGCAGTCTGGGGCTGAGCTGGTGGGGCCTGGGGCTTCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACACATTTACTGACTATGAAATGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAATGCATTGGAGCTATTGATCCTGAAACTGGTGGTACTGCCTACAATCAGAAGTTCAAGGGCAAGGCCATACTGACTGCAGACAAATCCTCTAGCACAGCCTACATGGAGCTCCGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTCTAACTGGGTTTGACTACTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA Heavy chain: Amino acid sequence (SEQ ID NO: 36):MEWSWVFLFLLSVIAGVQSQVQLQQSGAELVGPGASVTLSCKASGYTFTDYEMHWVKQTPVHGLECIGAIDPETGGTAYNQKFKGKAILTADKSSSTAYMELRSLTSEDSAVYYCLTGFDYWGQGTTLTVSSLight chain: DNA sequence (SEQ ID NO: 37):ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGTCTCAGTCATAATGTCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCTGGGGAGAAGGTCACCTTGACCTGCAGTGCCAGCTCAAGTGTGAATTCCAGCTACTTGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGATTTATAGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCTCTTATTTCTGCCATCAGTGGAGTAGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAALight chain: Amino acid sequence (SEQ ID NO: 38):MDFQVQIFSFLLISVSVIMSRGQIVLTQSPAIMSASPGEKVTLTCSASSSVNSSYLYWYQQKPGSSPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAASYFCHQWSSYPYTFGGGTKLEIK mAb 11B6Heavy chain: DNA sequence (SEQ ID NO: 39):ATGATGGTGTTAAGTCTTCTGTACCTGTTGACAGCCCTTCCGGGTATCCTGTCAGAGGTGCAGCTTCAGGAGTCAGGACCTGGCCTGGCAAAACCTTCTCAGACTCTGTCCCTCACCTGTTCTGTCACTGGCTACTCCATCACCAGTGATTACTGGAACTGGATCCGGAAATTCCCAGGGAATAAACTTGAATACATGGGGTACATAAACTACAGTGGTAACACTTACTACAATCCATCTCTCAAAAGTCGAATCTCCATAACTCGAGACACATCCAAGAACCAGTATTACCTGCAATTGAATTCTGTGACTACTGAGGACACAGCCACGTATTACTGTGCAAGATATGGGGGATTACGACAGGGTTCCTGGCACTTCGATGTCTGGGGCCCAGGGACCACGGTCACCGTCTCCTCAHeavy chain: Amino acid sequence (SEQ ID NO: 40):MMVLSLLYLLTALPGILSEVQLQESGPGLAKPSQTLSLTCSVTGYSITSDYWNWIRKFPGNKLEYMGYINYSGNTYYNPSLKSRISITRDTSKNQYYLQLNSVTTEDTATYYCARYGGLRQGSWHFDVWGPGTTVTVSSLight chain: DNA sequence (SEQ ID NO: 41):ATGGATTTTCAGGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATGTCCAGAGGACAAATTGTTCTCACCCAGTCTCCAGCAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATATCCTGCAGTGCCAGCTCAAGTGTAAGTTACATGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAAACCCTGGATTTATCGCACATCCAACCTGGCTTCTGGAGTCCCTGCGCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTATCATAGTTACCCAGCGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAALight chain: Amino acid sequence (SEQ ID NO: 42):MDFQVQIFSFLLISASVIMSRGQIVLTQSPAIMSASPGEKVTISCSASSSVSYMYWYQQKPGSSPKPWIYRTSNLASGVPARFSGSGSGTSYSLTISSMEAEDAATYYCQQYHSYPATFGGGTKLEIK mAb 11D3Heavy chain: DNA sequence (SEQ ID NO: 43):ATGGGTTGGCTGTGGAACTTGCTATTCCTGATGGCAGCTGCCCAAAGTGCCCAAGCACAGATCCAGTTGGTACAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATATCTTCACAACCTATGGAATGTACTGGGTGAAACAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACCTACTCTGGAGTGCCAACATATGTTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACATCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGTAGTTGCCGGGTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA Heavy chain: Amino acid sequence (SEQ ID NO: 44):MGWLWNLLFLMAAAQSAQAQIQLVQSGPELKKPGETVKISCKASGYIFTTYGMYWVKQAPGKGLKWMGWINTYSGVPTYVDDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCVVAGWFAYWGQGTLVTVSALight chain: DNA sequence (SEQ ID NO: 45):ATGGACATGAGGACCCCTGCTCAGTTTCTTGGAATCTTGTTGCTCTGGTTTCCAGGTATCAAATGTGACATCAAGATGACCCAGTCTCCATCTTCCATGTATGCATCTCTAGGAGAGAGAGTCACTATCACTTGCAAGGCGAGTCAGGACATTAAGAGCTATTTAAGCTGGTTCCAGCAGAAACCAGGGAAATCTCCTAAGACCCTGATCTATCGTGCAAATATATTGATAGATGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGCAAGATTATTCTCTCACCATCAGCAGCCTGGAGTATGAAGATATGGGAATTTATTATTGTCTACAATATGATGAGTTTCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAALight chain: Amino acid sequence (SEQ ID NO: 46):MDMRTPAQFLGILLLWFPGIKCDIKMTQSPSSMYASLGERVTITCKASQDIKSYLSWFQQKPGKSPKTLIYRANILIDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYCLQYDEFPYTFGGGTKLEIK mAb 11H2Heavy chain: DNA sequence (SEQ ID NO: 105)ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGACGTGAAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGACTGGAGTGGGTCGCATATGTTAGTAGTGGTGGTGGTAGTATCTATTATCCAGACACTGTAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTATTTGCAAATGAGCCGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGGCATGGGTCCCCCTTCGGTAGTAGCCGAGGGGCCTGGTTTGCTTACTGGGGCCAGGGGACTCTGGTCACTGTCTCTGCAHeavy chain: Amino acid sequence (SEQ ID NO: 106)MNLGLSLIFLVLVLKGVQCDVKLVESGGGLVQPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVAYVSSGGGSIYYPDTVKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARHGSPFGSSRGAWFAYWG QGTLVTVSALight chain: DNA sequence (SEQ ID NO: 107)ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGTGACATCCAGATGACTCAGTCTCCAGCCTCCCTGTCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGAGGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAATTATTACTGTCAACATTATGATGGTTTTCCGTTCACGTTCGGTGGTGGGACCAAGCTGGAGCTGAAALight chain: Amino acid sequence (SEQ ID NO: 108)MSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGNYYCQHYDGFPFTFGGGTKLELK mAb 6C12Heavy chain: DNA sequence (SEQ ID NO: 109)ATGGGTTGGCTGTGGAACTTGCTATTCCTGATGGCAGCTGCCCAAAGTGCCCAAGCACAGATCCAGTTGATACAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGGGTATACCTTCACAACCTTTGGAATGAGCTGGGTGAAACAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAGCACCTACTCTGGAGTGCCAACATATGCTGATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTTCATATTTCTGTGCAAGACACACCTTCCAAAGTCGCGGGTTGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAHeavy chain: Amino acid sequence (SEQ ID NO: 110)MGWLWNLLFLMAAAQSAQAQIQLIQSGPELKKPGETVKISCKASGYTFTTFGMSWVKQAPGKGLKWMGWISTYSGVPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTASYFCARHTFQSRGLAYWGQGTLVTVSALight chain: DNA sequence (SEQ ID NO: 111)ATGGGCATCAAAATGGAGTCACAGATTCAGGTCTTTGTATTCGTGTTTCTCTGGTTGTCTGGTGTTGACGGAGACATTGTGATGACCCAGTCTCACAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGCATCACCTGCAAGGCCAGTCAGGATGTGATTACTACTGTAGCCTGGTATCAACAGAAACCAGGACAATCTCCTAAACTACTGATTTACTCGGCATCCTACCGGTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACGGATTTCACTTTCACCATCACCAGTGTGCAGACTGAAGACCTGGCAGTTTATTACTGTCAGCAACATTATAGTACTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA Light chain: Amino acid sequence (SEQ ID NO: 112)MGIKMESQIQVFVFVFLWLSGVDGDIVMTQSHKFMSTSVGDRVSITCKASQDVITTVAWYQQKPGQSPKLLIYSASYRYTGVPDRFTGSGSGTDFTFTITSVQTEDLAVYYCQQHYSTPWTFGGGTKLEIK mAb 4H3Heavy chain: DNA sequence (SEQ ID NO: 113)ATGGGATGGAGCTGTATCATGCTCTTCTTGGCAGCAACAGCTACAGGTGTCCACTCCCAGGTCCAACTGCAGCAGCCTGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAACTACTGGATACACTGGATGAAGCAGAGGCCTGGACGAGGCCTTGAGTGGATTGGAAGGATTGATCCTAATAGTGGTGGTACTAAGTACAATGAGAAGTTCAAGAGCAAGGCCACACTGACTGTCGACAAACCCTCCATCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTATTGTGCAGCATTCGGTAGTACCTACGGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA Heavy chain: Amino acid sequence (SEQ ID NO: 114)MGWSCIMLFLAATATGVHSQVQLQQPGAELVKPGASVKLSCKASGYTFTNYWIHWMKQRPGRGLEWIGRIDPNSGGTKYNEKFKSKATLTVDKPSITAYMQLSSLTSEDSAVYYCAAFGSTYGFAYWGQGTLVTVSALight chain: DNA sequence (SEQ ID NO: 115)ATGGATTCACAGGCCCAGGTTCTTATATTGCTGCTGCTATGGGTATCTGGTACCTGTGGGGACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGTTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAA Light chain: Amino acid sequence (SEQ ID NO: 116)MDSQAQVLILLLLWVSGTCGDIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLYTFGGGTKLEIK mAb 4D8Heavy chain: DNA sequence (SEQ ID NO: 117)ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAAGTGACGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTGACTATTACATGTATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAGTCCTGGTGGTGGTAGCACCTATTATCCGGACACTATAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCCGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTACAAGACATGGGTCCCCCTACGGTAGTAGTCGAGGGGCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAHeavy chain: Amino acid sequence (SEQ ID NO: 118)MNLGLSLIFLVLVLKGVQCEVTLVESGGGLVQPGGSLKLSCAASGFTFSDYYMYWVRQTPEKRLEWVAYISPGGGSTYYPDTIKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCTRHGSPYGSSRGAWFAYWGQGTLVTVSALight chain: DNA sequence (SEQ ID NO: 119)ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGTGACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGGAAAAACCTTAGCAGAAGGTGTGCCAGCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTACAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATGATGGTATTCCGGTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAALight chain: Amino acid sequence (SEQ ID NO: 120)MSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNGKTLAEGVPARFSGSGSGTQFSLKINSLQPEDFGSYYCQHHDGIPVTFGAGTKLELK mAb 2E2Heavy chain: DNA sequence (SEQ ID NO: 121)ATGAACTTGGGGCTCAGCTTGATTTTCCTTGTCCTTGTTTTAAAAGGTGTCCAGTGTGAAGTGAAGCTGGTGGAGTCGGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGTAGCCTCTGGATTCACTTTCAGTGACTATCACATGCATTGGGTTCGCCAGACTCCAGAGAAGAGGCTGGAGTGGGTCGCATACATTAGTAAAGGTGGTGGTAGCACCTATTATCCAGACACTGAAAAGGGCCGATTCACCATCTCCAGAGACAATGCCAAGAATACCCTGTACCTGCAAATGAGCCGTCTGAAGTCTGAGGACACAGCCATGTATTACTGTGCAAGATCCCCCGGCCCTAGTAGCTTCTACTGGTACTTCGATGTCTGGGGCACAGGGACCACGGTCACCGTCTCCTCA Heavy chain: Amino acid sequence (SEQ ID NO: 122)MNLGLSLIFLVLVLKGVQCEVKLVESGGGLVQPGGSLKLSCVASGFTFSDYHMHWVRQTPEKRLEWVAYISKGGGSTYYPDTEKGRFTISRDNAKNTLYLQMSRLKSEDTAMYYCARSPGPSSFYWYFDVWGTGTTVTVSS Light chain: DNA sequence (SEQ ID NO: 123)ATGAGTGTGCCCACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGGTGCCAGATGTGACATCCAGATGACTCAGTCTCCAGCCTCCCTATCTGCATCTGTGGGAGAAACTGTCACCATCACATGTCGAGCAAGTGAGAATATTTACAGTTATTTAGCATGGTATCAGCAGAAACAGGGAAAATCTCCTCAGCTCCTGGTCTATAATGCAAAAACCTTAGCAGAAGGTGTGCCATCAAGGTTCAGTGGCAGTGGATCAGGCACACAGTTTTCTCTGAAGATCAACAGCCTGCAGCCTGAAGATTTTGGGAGTTATTACTGTCAACATCATTATGGTATTCCGGTCACGGTCGGTGTAGGGACCAAGCTGGAGCTGAAALight chain: Amino acid sequence (SEQ ID NO: 124)MSVPTQVLGLLLLWLTGARCDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKQGKSPQLLVYNAKTLAEGVPSRFSGSGSGTQFSLKINSLQPEDFGSYYCQHHYGIPVTVGVGTKLELK

Example 4 Construction of CHO Cell Line Expressing Canine IL-4 ReceptorAlpha Chain and Use in Ligand Blockade Assays

The gene encoding full length canine IL-4 receptor alpha chain(cIL-4R_(α); SEQ ID NO: 4) was synthesized and sub-cloned into amammalian expression vectors. The resulting plasmid was transfected intoCHO DG44 cells. At 48 hours post-transfection, the cells were dilutedinto 96-well plates to generate single cell clones. About 130 cloneswere obtained after a 4-week incubation. All of the clones were screenedfor expression of cIL-4R_(α) by FACS using the anti-cIL-4R_(α)monoclonal antibody 6B2. Three clones were selected for stabilityevaluation. Stability was monitored for 20 passages by FACS.

In order to assess the ability of monoclonal antibodies specific tocanine IL-4 receptor alpha to block the binding of canine IL-4 to canineIL-4 R alpha expressed on the surface of CHO cells, a ligand blockadeassay was set as follows:

Reagent and Equipments:

-   -   Cell growth medium: CD OptiCHO medium+8 mM L-Glutamine+0.018%        F-68    -   FACS Buffer: BD Pharmingen Stain Buffer (BD cat#: 554657)    -   R-phycoerythin conjugated Streptavidin (Life Technologies: SB66)    -   Canine IL-4 (R&D system, cat #754-CL/CF)    -   Lightning-Link Biotin Conjugation Kit Type A (Novus: 704-0010)        used to biotinylate canine IL-4 as per manufacturer's        recommendation    -   Flow cytometer: BD Accuri-C6

Procedure:

-   -   1. CHO-DH44-canIL-4R_(α) cell grown to 2-4×10⁶ cells/mL with        more than 96% viability.    -   2. The cells were spun down, the supernatant discarded, and the        cells were suspended in FACS buffer to 2×10⁷ cells/mL.    -   3. The cells were distributed into a U-shape 96-well plate, 50        μl each well.    -   4. The anti-canine IL-4Rα mAbs in FACS buffer was diluted        three-fold on a 96-well plate from top down to bottom well,        starting at 50 μg/mL.    -   5. 50 μl of each diluted Ab was transferred into the cell plate        and then incubated on ice for 30 min.    -   6. The cells were washed twice with FACS buffer.    -   7. The cells were resuspended into 100 μl of biotinylated canine        IL-4 at 0.32 μg/mL in FACS buffer and incubated on ice for 30        min.    -   8. The cells were washed twice with FACS buffer.    -   9. The cells were responded into 100 μl of R-phycoerythin        conjugated Streptavidin (1:1000 dilution) in FACS buffer and        incubated on ice for 30 min.    -   10. The cells were washed twice with FACS buffer.    -   11. The cells were brought up to 300 μl in FACS buffer.    -   12. 10,000 cells were read for each sample by BD Accuri-C6.    -   13. The resulting readout were analyzed by FlowJo to get the        mean fluorescent intensity (MFI).

A dose response curve for the binding of canine IL-4 to canine IL-4R_(α)expressed on the surface of CHO cells was obtained using the cell-basedCHO-cIL-4R_(α) binding assay (see, FIG. 2A). A half maximal effectiveconcentration (EC50) of 25 nM was determined from this curve. Next, doseresponse curves for the binding of CHO-cIL-4R_(α) by the mouseanti-canine IL-4R_(α) monoclonal antibodies (mAbs): 11B6, 4D8, 4H3, 2E2,11H2, and 6C12 were obtained (see, FIG. 2B). The half maximal effectiveconcentrations (EC50) for each of the antibodies is provided in Table 2below.

TABLE 2 Binding/Blocking of Various mABs mABs EC50 (nM) IC50 (nM) 11B67.5 53.2 4D8 1.1 4.2 4H3 1.6 3.9 2E2 1.2 2.1 11H2 1.2 1.7/1.0* 6C12 8.619.3 *Determinations from two separate studies

The mouse anti-canine IL-4R_(α) monoclonal antibodies (mAbs) were thenassayed for their ability to block the binding of canine IL-4 to thecell-based CHO-cIL-4R_(α). As depicted in FIG. 3A the five mAbs, 11B6,4D8, 4H3, 2E2, and 11H2 displayed significant blocking ability. In acomplementary study a sixth mAbs was tested (6C12), and compared withone of the five mAbs tested (11H2) in FIG. 3A. As is apparent from FIG.3B and Table 2, 6C12 mAbs has a significantly higher half maximalinhibitory concentration (IC50) than the 11H2 mAbs. Four of anti-cIL-4Rαmonoclonal antibodies, 4D8, 2E2, 4D8, and 11H2 showed superior blockingability, as can be seen in FIGS. 3A and 3B, as well as in Table 2.

Example 5 Amino Acid Sequences of the Mouse CDRS

CDRs from mouse anti-canine IL-4 receptor α chain monoclonal antibodies:

SEQ ID NO: VL CDR-1 1A3 Arg Ala Ser Ser Ser Val Ser Phe Met Phe  47 1A9Lys Ala Ser Gln Asn Val Arg Ser Ala Val Ala  48 1B12Arg Ser Ser Lys Ser Leu Leu His Ser Asn Gly Asn Thr Tyr Leu Phe  4910C12Lys Ser Ser Gln Ser Leu Leu Asn Ser Ser Asn Gln Lys Asn Tyr Leu Ala  5010F2 Lys Ser Ser Gln Asn Leu Leu Tyr Gly Gly Asn Gln Lys Asn Tyr Leu Ala 51 10E10 Lys Ala Ser Asp His Ile Asn Asn Trp Leu Ala  52 10G8Ser Ala Ser Ser Ser Val Asn Ser Ser Tyr Leu Tyr  53 11B6Ser Ala Ser Ser Ser Val Ser Tyr Met Tyr  54 11D3Lys Ala Ser Gln Asp Ile Lys Ser Tyr Leu Ser  55 11H2Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 129 6C12Lys Ala Ser Gln Asp Val Ile Thr Thr Val Ala 130 4D8Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 129 4H3Lys Ser Ser Gln Ser Leu Leu Asn Ser Arg Thr Arg Lys Asn Tyr Leu Ala 1312E2 Arg Ala Ser Glu Asn Ile Tyr Ser Tyr Leu Ala 129 VL CDR-2 1A3Asp Thr Ser Asn Leu Ala Ser  56 1A9 Leu Ala Ser Asn Arg His Thr  57 1B12Arg Met Ser Asn Leu Ala Ser  58 10C12 Phe Ala Ser Thr Arg Glu Ser  5910F2 Trp Ala Ser Thr Arg Glu Ser  60 10E10 Gly Ala Thr Ser Leu Glu Thr 61 10G8 Ser Thr Ser Asn Leu Ala Ser  62 11B6Arg Thr Ser Asn Leu Ala Ser  63 11D3 Arg Ala Asn Ile Leu Ile Asp  6411H2 Asn Ala Lys Thr Leu Ala Glu 132 6C12 Ser Ala Ser Tyr Arg Tyr Thr133 4D8 Asn Gly Lys Thr Leu Ala Glu 134 4H3 Trp Ala Ser Thr Arg Glu Ser 60 2E2 Asn Ala Lys Thr Leu Ala Glu 132 VL CDR-3 1A3Gln Gln Trp Ser Ser Asn Pro Leu Thr  65 1A9Leu Gln His Trp Asn Tyr Pro Phe Thr  66 1B12Met Gln His Leu Glu Tyr Pro Phe Thr  67 10C12Gln Gln His Tyr Ser Thr Pro Tyr Thr  68 10F2Gln Gln Tyr Tyr Asp Tyr Pro Tyr Thr  69 10E10His Gln Tyr Trp Ser Ile Pro Tyr Thr  70 10G8His Gln Trp Ser Ser Tyr Pro Tyr Thr  71 11B6Gln Gln Tyr His Ser Tyr Pro Ala Thr  72 11D3Leu Gln Tyr Asp Glu Phe Pro Tyr Thr  73 11H2Gln His Tyr Asp Gly Phe Pro Phe Thr 135 6C12Gln Gln His Tyr Ser Thr Pro Trp Thr 136 4D8Gln His His Asp Gly Ile Pro Val Thr 137 4H3Lys Gln Ser Tyr Asn Leu Tyr Thr 138 2E2Gln His His Tyr Gly Ile Pro Val Thr 139 VH CDR-1 1A3 Asp Phe Gly Met His 74 1A9 Ser Ser Trp Met Asn  75 1B12 Asp Tyr Tyr Met Asn  76 10C12Asp Tyr Glu Met His  77 10F2 Ser Tyr Gly Val Ser  78 10E10Thr Tyr Asp Ile His  79 10G8 Asp Tyr Glu Met His  80 11B6Ser Asp Tyr Trp Asn  81 11D3 Thr Tyr Gly Met Tyr  82 11H2Asp Tyr Tyr Met Tyr 140 6C12 Thr Phe Gly Met Ser 141 4D8Asp Tyr Tyr Met Tyr 140 4H3 Asn Tyr Trp Ile His 142 2E2Asp Tyr His Met His 143 VH CDR-2 1A3Tyr Ile Ser Ser Gly Ser Gly Thr Ile Tyr Tyr Ala Asp Thr Val Arg Gly  831A9 Arg Ile Tyr Pro Gly Asp Gly Asp Thr Lys Tyr Asn Gly Lys Phe Lys Gly 84 1B12Asp Ile Ile Pro Ser Asn Gly Gly Thr Ser Tyr Asn Gln Lys Phe Lys Gly  8510C12Ala Ile Asp Pro Glu Thr Cys Gly Thr Ala Tyr Asn Gln Lys Phe Lys Gly  8610F2 Val Ile Trp Gly Asp Gly Ser Thr Tyr Phe His Ser Ala Leu Ile Ser  8710E10Trp Ile Tyr Pro Arg Asp Gly Arg Thr Thr Tyr Asn Glu Lys Phe Lys Ala  8810G8 Ala Ile Asp Pro Glu Thr Gly Gly Thr Ala Tyr Asn Gln Lys Phe Lys Gly 89 11B6 Tyr Ile Asn Tyr Ser Gly Asn Thr Tyr Tyr Asn Pro Ser Leu Lys Ser 90 11D3Trp Ile Asn Thr Tyr Ser Gly Val Pro Thr Tyr Val Asp Asp Phe Lys Gly  9111H2 Tyr Val Ser Ser Gly Gly Gly Ser Ile Tyr Tyr Pro Asp Thr Val Lys Gly144 6C12Trp Ile Ser Thr Tyr Ser Gly Val Pro Thr Tyr Ala Asp Asp Phe Lys Gly 1454D8 Tyr Ile Ser Pro Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Ile Lys Gly146 4H3Arg Ile Asp Pro Asn Ser Gly Gly Thr Lys Tyr Asn Glu Lys Phe Lys Ser 1472E2 Tyr Ile Ser Lys Gly Gly Gly Ser Thr Tyr Tyr Pro Asp Thr Glu Lys Gly148 VH CDR-3 1A3 Gly Asp Leu Tyr Tyr Gly Ser Ser Phe Asp Ala Tyr  92 1A9Asp Asp Tyr Asp Trp Ala Ser  93 1B12Gly Ile Ser Tyr Tyr Gly Asn Arg Tyr Tyr Phe Thr Met Asp Tyr  94 10C12Ser Lys Leu Gly Arg Gly Trp Tyr Phe Asp Val  95 10F2Gln Gly Thr Ile Tyr Asp Gly Tyr Tyr Asn Tyr Ala Met Asp Tyr  96 10E10Ser Ser Pro Phe Gly Tyr  97 10G8 Gly Phe Asp Tyr  98 11B6Tyr Gly Gly Leu Arg Gln Gly Ser Trp His Phe Asp Val  99 11D3Ala Gly Trp Phe Ala Tyr 100 11H2His Gly Ser Pro Phe Gly Ser Ser Arg Gly Ala Trp Phe Ala Tyr 149 6C12His Thr Phe Gln Ser Arg Gly Leu Ala Tyr 150 4D8His Gly Ser Pro Tyr Gly Ser Ser Arg Gly Ala Trp Phe Ala Tyr 151 4H3Phe Gly Ser Thr Tyr Gly Phe Ala Tyr 152 2E2Ser Pro Gly Pro Ser Ser Phe Tyr Trp Tyr Phe Asp Val 153

TABLE 3 CANONICAL STRUCTURES L1 L2 L3 H1 H2 H3 1A3 L1-1 L2-1 L3-1 H1-1H2-3A H3-12 1A9 L1-2A L2-1 L3-1 H1-1 H2-2A H3-7 1B12 L1-4 L2-1 L3-1 H1-1H2-2B H3-15 10C12 L1-3 L2-1 L3-1 H1-1 * H3-11 10F2 L1-3 L2-1 L3-1 H1-1H2-1 H3-15 10E10 L1-2A L2-1 L3-1 H1-1 H2-2B H3-6 10G8 L1-6 L2-1 L3-1H1-1 H2-2B H3-4 11B6 L1-1 L2-1 L3-1 H1-1 H2-1 H3-13 11D3 L1-2A L2-1 L3-1H1-1 H2-2A** H3-6 11H2 L1-6 L2-1 L3-1 H1-1 H2-3A H3-15 6C12 L1-6 L2-1L3-1 H1-1 H2-2A H3-10 4D8 L1-6 L2-1 L3-1 H1-1 H2-3A H3-15 4H3 L1-3 L2-1L3-3 H1-1 H2-3A H3-9 2E2 L1-6 L2-1 L3-1 H1-1 H2-3A H3-13 * Cysteine inthe CDR **The best assignment that could be made in view of theparticular pattern.

Example 6 Epitope Mapping of Murine Anti-Canine IL-4 Receptor AlphaAntibodies

The interaction of antibodies with their cognate protein antigens ismediated through the binding of specific amino acids of the antibodies(paratopes) with specific amino acids (epitopes) of target antigens. Anepitope is an antigenic determinant that causes a specific reaction byan immunoglobulin. An epitope consists of a group of amino acids on thesurface of the antigen. A protein of interest may contain severalepitopes that are recognized by different antibodies. The epitopesrecognized by antibodies are classified as linear or conformationalepitopes. Linear epitopes are formed by a stretch of a continuoussequence of amino acids in a protein, while conformational epitopes arecomposed of amino acids that are discontinuous (e.g., far apart) in theprimary amino acid sequence, but are brought together uponthree-dimensional protein folding.

Epitope mapping refers to the process of identifying the amino acidsequences (i.e., epitopes) that are recognized by antibodies on theirtarget antigens. Identification of epitopes recognized by monoclonalantibodies (mAbs) on target antigens has important applications. Forexample, it can aid in the development of new therapeutics, diagnostics,and vaccines. Epitope mapping can also aid in the selection of optimizedtherapeutic mAbs and help elucidate their mechanisms of action. Epitopeinformation on IL-4 receptor alpha can also elucidate unique epitopes,and define the protective or pathogenic effects of vaccines. Epitopeidentification also can lead to development of subunit vaccines based onchemical or genetic coupling of the identified peptide epitope to acarrier protein or other immunostimulating agents.

Epitope mapping can be carried out using polyclonal or monoclonalantibodies and several methods are employed for epitope identificationdepending on the suspected nature of the epitope (i.e., linear versusconformational). Mapping linear epitopes is more straightforward andrelatively, easier to perform. For this purpose, commercial services forlinear epitope mapping often employ peptide scanning. In this case, anoverlapping set of short peptide sequences of the target protein arechemically synthesized and tested for their ability to bind antibodiesof interest. The strategy is rapid, high-throughput, and relativelyinexpensive to perform. On the other hand, mapping of a discontinuousepitope is more technically challenging and requires more specializedtechniques such as x-ray co-crystallography of a monoclonal antibodytogether with its target protein, Hydrogen-Deuterium (H/D) exchange,Mass Spectrometry coupled with enzymatic digestion as well as severalother methods known to those skilled in the art.

Mapping of Canine IL-4 Receptor Alpha Epitopes Using Mass Spectroscopy:

A method based on chemical crosslinking and mass spectrometry detectionwas employed to identify epitopes recognized by anti-canine IL-4receptor alpha mAbs [CovalX Instrument Incorporated]. The application ofthis technology to epitope mapping of canine IL-4 receptor alpha chainresulted in identification of epitopes recognized by the mAbs listed inTable 4.

The results from the epitope mapping of canine IL-4 receptor alpha withthe six antibodies included in Table 4. indicates that the mAbsrecognize specific peptide epitopes that are present within theextracellular domain of canine IL-4 receptor alpha. Notably, two tothree epitopes were identified for each of the six monoclonal antibodies(mAbs) tested. Interestingly, one of the epitopes identified for mAbs2E2 was found to have the exact same amino acid sequence as that formAbs 11B6 (i.e., SEQ ID NO: 158). As depicted in Table 4 below, mAbs:4D8, 11H2, and 11B6 all recognize an epitope, labeled with a “¹” that isa portion of the same linear amino acid sequence; mAbs: 11E2, 4H3, and2E2 all recognize an epitope labeled with a “²” that is a portion ofanother linear amino acid sequence; and mAbs 4H3 and 2H2 recognize anepitope labeled with a “³” that is a portion of a third linear aminoacid sequence. This relative consistency in the identification of therelevant epitopes indicates that these six monoclonal antibodiesrecognize a limited number of portions of canine IL-4 receptor alpha,within its extracellular domain.

TABLE 4 IL-4 RECEPTOR ALPHA EPITOPESRECOGNIZED BY ANTI-CANINE IL-4 RECEPTOR ALPHA MONOCLONAL ANTIBODIES SEQANTIBODY ID NO: EPITOPE SEQUENCE 4D8 125 SAELRLSYQLD 126 FQPSKHVKPRT ¹11H2 127 AGQQLLWSGSFQPSKHVKPRT ¹ 128 TLKSGASYS ² 4H3 154 EDSVCVCSMPI ³155 MWTNPYPTENHL 156 ASTLKSG ² 11B6 157 WSGSFQPSKHVKPR ¹ 158VYNVTYMGPTLR 2E2 159 VLHEPSCFSDYISTSVCQ 160 ENREDSVCVCSMPI ³ 161KSGASYSARVRAW² 6C12 158 VYNVTYMGPTLR 162 YYEPWEQHLP ^(1,2,3)identifythree individual groups of epitopes arising from three portions of theantigen.

Together with the CDRs provided in Example 5 for the six antibodieslisted in Table 4 above, a one to one relationship is defined betweeneach set of CDRs and their corresponding epitopes in Table 4. Thisrelationship allows a defined linkage between the set of 6 CDRs inExample 5 for each of the six antibodies in Table 4 and thecorresponding epitopes that they bind. Accordingly, antibodies (e.g.,caninized antibodies) with the defined set of 6 CDRs provided in Example5 that bind corresponding epitopes in Table 4 are also part of thepresent invention.

Example 7 Construction of Caninized Anti-Canine IL-4 Receptor AlphaMonoclonal Antibodies

In order to execute the process of caninization, the DNA sequence thatencodes the heavy and light chains of canine IgG were determined. TheDNA and protein sequence of the canine heavy and light chains are knownin the art and can be obtained by searching of the NCBI gene and proteindatabases. As indicated above, for canine antibodies there are fourknown IgG subtypes: IgG-A, IgG-B, IgG-C, and IgG-D, and two types oflight chains, i.e., kappa and lambda. Without being bound by anyspecific approach, the overall process of producing caninized heavy andlight chains that can be mixed in different combinations to producecaninized anti-canine IL-4 receptor alpha mAbs involves the followingscheme:

i) Identify the DNA sequence of VH and VL domains comprising the CDRs ofdesired anti-IL-4 receptor alpha mAbs

ii) Identify the H and L chain CDRs of desired anti-IL-4 receptor mAbs

iii) Identify a suitable sequence for H and L chain of canine IgG

iv) Identify the DNA sequence encoding the endogenous CDRs of canine IgGH and L chains of the above sequence.

v) Replace the DNA sequence encoding endogenous canine H and L chainCDRs with DNA sequences encoding the desired anti-IL-4 receptor alphaCDRs. In addition, optionally replace some canine framework residueswith selected residues from the desired anti-IL-4 receptor mAb frameworkregions.

vi) Synthesize the DNA from step (v), clone it into a suitableexpression plasmid, and transfect the plasmids containing desiredcaninized H and L chains into HEK 293 cells. vii) Purify expressedcaninized antibody from HEK 293 supernatant.

viii) Test purified caninized antibody for binding to canine IL-4receptor alpha chain.

The application of the above outlined steps resulted in a set ofcaninized H and L chain sequences for which the SEQ ID NOs. are listedin Table 5 below.

TABLE 5 CANINIZED FULL-LENGTH HEAVY AND LIGHT CHAIN SEQUENCES H chain orL chain Nucleic Acid Amino Acid vH1 SEQ ID NO: 163 SEQ ID NO: 164 vH2SEQ ID NO: 165 SEQ ID NO: 166 vH3 SEQ ID NO: 167 SEQ ID NO: 168 vL1 SEQID NO: 169 SEQ ID NO: 170 vL2 SEQ ID NO: 171 SEQ ID NO: 172 vL3 SEQ IDNO: 173 SEQ ID NO: 174

The present invention provides caninized antibodies formed by thecombination of various caninized heavy and light chains listed in theTable 5 above; such antibodies have particularly tight binding withcanine IL-4 receptor alpha. In a particular embodiment the heavy chaincomprises the amino acid sequence of SEQ ID NO: 164 and the light chaincomprises the amino acid sequence of SEQ ID NO: 170. In a moreparticular embodiment of this type, the heavy chain is encoded by thenucleotide sequence of SEQ ID NO: 163 and the light chain is encoded bythe nucleotide sequence of SEQ ID NO: 169. In another embodiment theheavy chain comprises the amino acid sequence of SEQ ID NO: 166 and thelight chain comprises the amino acid sequence of SEQ ID NO: 172. In amore particular embodiment of this type, the heavy chain is encoded bythe nucleotide sequence of SEQ ID NO: 165 and the light chain is encodedby the nucleotide sequence of SEQ ID NO: 171. In still anotherembodiment the heavy chain comprises the amino acid sequence of SEQ IDNO: 168 and the light chain comprises the amino acid sequence of SEQ IDNO: 174. In a more particular embodiment of this type, the heavy chainis encoded by the nucleotide sequence of SEQ ID NO: 167 and the lightchain is encoded by the nucleotide sequence of SEQ ID NO: 173. Bindingstudies to IL-4 receptor alpha by these caninized antibodies aredepicted in FIG. 4, as described in Example 8, below.

As indicated above, the Fc portion of the caninized antibodies is basedon modified sequences of canine IgG-B in order to remove ADCC and CDCeffector functions. The Fc regions of these antibodies may be replacedwith a modified Fc from other canine IgG isotypes and/or can be combinedwith substitute hinge regions as discussed above, and exemplified anddisclosed in U.S. provisional application 62/030,812 filed Jul. 30,2014; U.S. provisional application 62/057,541 filed Sep. 30, 2014; U.S.provisional application 62/092,496 filed Dec. 16, 2014; U.S. provisionalapplication 62/172,511, filed Jun. 8, 2015; and WO 2015/091910, thecontents of all of which are hereby incorporated by reference in theirentireties.

CANINZED 4H3 (vH1) SEQ ID NO: 163GAGGTGCAGCTGGTGGAGAGCGGAGGCGACCTGGTGAAACCCGGAGGCAGCCTGAGACTGAGCTGTGTGGCCAGCGGCTACACCTTCACCAACTACTGGATTCATTGGGTGAGGCAGGCTCCCGGCAAAGGACTGCAGTGGGTGGCCAGGATTGATCCCAACAGCGGCGGCACCAAGTACAACGAGAAGTTCAAGAGCAGGTTCACCATCAGCAGGGACAACGCCAAGAACACCCTCTACCTGCAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCACCAGGTTCGGCAGCACCTACGGCTTCGCCTACTGGGGCCAAGGCACCCTGGTGACCGTGAGCAGCGCTTCCACAACCGCGCCATCAGTCTTTCCGTTGGCCCCATCATGCGGGTCGACGAGCGGATCGACTGTGGCCCTGGCGTGCTTGGTGTCGGGATACTTTCCCGAACCCGTCACGGTCAGCTGGAACTCCGGATCGCTTACGAGCGGTGTGCATACGTTCCCCTCGGTCTTGCAATCATCAGGGCTCTACTCGCTGTCGAGCATGGTAACGGTGCCCTCATCGAGGTGGCCCTCCGAAACGTTCACATGTAACGTAGCACATCCAGCCTCCAAAACCAAGGTGGATAAACCCGTGCCGAAAAGAGAGAATGGGCGGGTGCCTCGACCCCCTGATTGCCCCAAGTGTCCGGCTCCGGAAATGCTCGGTGGACCCTCAGTGTTTATCTTCCCTCCGAAGCCCAAGGACACTCTGCTGATCGCGCGCACTCCAGAAGTAACATGTGTAGTGGTGGCACTTGATCCCGAGGACCCCGAAGTCCAGATCTCCTGGTTTGTAGATGGGAAACAGATGCAGACCGCAAAAACTCAACCCAGAGAGGAGCAGTTCGCAGGAACATACCGAGTGGTATCCGTCCTTCCGATTGGCCACCAGGACTGGTTGAAAGGGAAGCAGTTTACGTGTAAAGTCAACAATAAGGCGTTGCCTAGCCCTATTGAGCGGACGATTTCGAAAGCTAGGGGACAGGCCCACCAGCCATCGGTCTATGTCCTTCCGCCTTCCCGCGAGGAGCTCTCGAAGAATACAGTGAGCCTTACATGCCTCATTAAGGATTTCTTCCCGCCTGATATCGACGTAGAGTGGCAATCAAACGGTCAACAGGAGCCGGAATCCAAGTATAGAACCACTCCGCCCCAGCTTGACGAGGACGGATCATACTTTTTGTATTCAAAACTGTCGGTGGATAAGAGCCGGTGGCAGAGAGGTGACACCTTCATCTGTGCGGTGATGCACGAAGCACTCCATAATCACTACACCCAAGAGAGCCTCTCGCATTCCCCCGGAAAG SEQ ID NO: 164EVQLVESGGDLVKPGGSLRLSCVASGYTFTNYWIHWVRQAPGKGLQWVARIDPNSGGTKYNEKFKSRFTISRDNAKNTLYLQMNSLRAEDTAVYYCTRFGSTYGFAYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGKCANINZED 4H3 (vH2) SEQ ID NO: 165GAGGTGCAGCTGGTGGAGAGCGGCGGAGATCTGGTGAAGCCCGGCGGAAGCCTGAGACTGAGCTGTGTGGCCAGCGGCTACACCTTCACCAACTACTGGATTCATTGGGTGAGACAGGCCCCTGGCAAGGGCCTGCAGTGGATCGGCAGGATCGACCCCAACAGCGGCGGCACCAAGTACAACGAGAAGTTCAAGAGCAAGGCCACCCTGAGCGTGGACAAGGCCAAGAACACCCTGTACCTGCAGATGAACTCCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCGCCTTTGGCAGCACCTACGGCTTCGCCTACTGGGGCCAGGGAACCCTGGTGACCGTGAGCAGCGCTTCCACAACCGCGCCATCAGTCTTTCCGTTGGCCCCATCATGCGGGTCGACGAGCGGATCGACTGTGGCCCTGGCGTGCTTGGTGTCGGGATACTTTCCCGAACCCGTCACGGTCAGCTGGAACTCCGGATCGCTTACGAGCGGTGTGCATACGTTCCCCTCGGTCTTGCAATCATCAGGGCTCTACTCGCTGTCGAGCATGGTAACGGTGCCCTCATCGAGGTGGCCCTCCGAAACGTTCACATGTAACGTAGCACATCCAGCCTCCAAAACCAAGGTGGATAAACCCGTGCCGAAAAGAGAGAATGGGCGGGTGCCTCGACCCCCTGATTGCCCCAAGTGTCCGGCTCCGGAAATGCTCGGTGGACCCTCAGTGTTTATCTTCCCTCCGAAGCCCAAGGACACTCTGCTGATCGCGCGCACTCCAGAAGTAACATGTGTAGTGGTGGCACTTGATCCCGAGGACCCCGAAGTCCAGATCTCCTGGTTTGTAGATGGGAAACAGATGCAGACCGCAAAAACTCAACCCAGAGAGGAGCAGTTCGCAGGAACATACCGAGTGGTATCCGTCCTTCCGATTGGCCACCAGGACTGGTTGAAAGGGAAGCAGTTTACGTGTAAAGTCAACAATAAGGCGTTGCCTAGCCCTATTGAGCGGACGATTTCGAAAGCTAGGGGACAGGCCCACCAGCCATCGGTCTATGTCCTTCCGCCTTCCCGCGAGGAGCTCTCGAAGAATACAGTGAGCCTTACATGCCTCATTAAGGATTTCTTCCCGCCTGATATCGACGTAGAGTGGCAATCAAACGGTCAACAGGAGCCGGAATCCAAGTATAGAACCACTCCGCCCCAGCTTGACGAGGACGGATCATACTTTTTGTATTCAAAACTGTCGGTGGATAAGAGCCGGTGGCAGAGAGGTGACACCTTCATCTGTGCGGTGATGCACGAAGCACTCCATAATCACTACACCCAAGAGAGCCTCTCGCATTCCCCCGGAAAG SEQ ID NO: 166EVQLVESGGDLVKPGGSLRLSCVASGYTFTNYWIHWVRQAPGKGLQWIGRIDPNSGGTKYNEKFKSKATLSVDKAKNTLYLQMNSLRAEDTAVYYCAAFGSTYGFAYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGKCANINZED 4H3 (vH3): SEQ ID NO: 167GAGGTGCAGCTGGTGGAGAGCGGCGGCGATCTGGTGAAGCCTGGCGGAAGCCTGAGACTGAGCTGCGTGGCCAGCGGCTACACCTTCACCAACTACTGGATTCATTGGATGAGGCAGGCCCCTGGCAAGGGACTGCAGTGGATCGGCAGAATCGACCCCAACAGCGGCGGCACCAAGTACAACGAGAAGTTCAAGAGCAAGGCCACCCTGAGCGTGGACAAGGCCAAGAACACCGCCTACATGCAGCTGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCGCCGCCTTTGGCAGCACCTACGGCTTCGCCTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCTTCCACAACCGCGCCATCAGTCTTTCCGTTGGCCCCATCATGCGGGTCGACGAGCGGATCGACTGTGGCCCTGGCGTGCTTGGTGTCGGGATACTTTCCCGAACCCGTCACGGTCAGCTGGAACTCCGGATCGCTTACGAGCGGTGTGCATACGTTCCCCTCGGTCTTGCAATCATCAGGGCTCTACTCGCTGTCGAGCATGGTAACGGTGCCCTCATCGAGGTGGCCCTCCGAAACGTTCACATGTAACGTAGCACATCCAGCCTCCAAAACCAAGGTGGATAAACCCGTGCCGAAAAGAGAGAATGGGCGGGTGCCTCGACCCCCTGATTGCCCCAAGTGTCCGGCTCCGGAAATGCTCGGTGGACCCTCAGTGTTTATCTTCCCTCCGAAGCCCAAGGACACTCTGCTGATCGCGCGCACTCCAGAAGTAACATGTGTAGTGGTGGCACTTGATCCCGAGGACCCCGAAGTCCAGATCTCCTGGTTTGTAGATGGGAAACAGATGCAGACCGCAAAAACTCAACCCAGAGAGGAGCAGTTCGCAGGAACATACCGAGTGGTATCCGTCCTTCCGATTGGCCACCAGGACTGGTTGAAAGGGAAGCAGTTTACGTGTAAAGTCAACAATAAGGCGTTGCCTAGCCCTATTGAGCGGACGATTTCGAAAGCTAGGGGACAGGCCCACCAGCCATCGGTCTATGTCCTTCCGCCTTCCCGCGAGGAGCTCTCGAAGAATACAGTGAGCCTTACATGCCTCATTAAGGATTTCTTCCCGCCTGATATCGACGTAGAGTGGCAATCAAACGGTCAACAGGAGCCGGAATCCAAGTATAGAACCACTCCGCCCCAGCTTGACGAGGACGGATCATACTTTTTGTATTCAAAACTGTCGGTGGATAAGAGCCGGTGGCAGAGAGGTGACACCTTCATCTGTGCGGTGATGCACGAAGCACTCCATAATCACTACACCCAAGAGAGCCTCTCGCATTCCCCCGGAAAG SEQ ID NO: 168EVQLVESGGDLVKPGGSLRLSCVASGYTFTNYWIHWMRQAPGKGLQWIGRIDPNSGGTKYNEKFKSKATLSVDKAKNTAYMQLNSLRAEDTAVYYCAAFGSTYGFAYWGQGTLVTVSSASTTAPSVFPLAPSCGSTSGSTVALACLVSGYFPEPVTVSWNSGSLTSGVHTFPSVLQSSGLYSLSSMVTVPSSRWPSETFTCNVAHPASKTKVDKPVPKRENGRVPRPPDCPKCPAPEMLGGPSVFIFPPKPKDTLLIARTPEVTCVVVALDPEDPEVQISWFVDGKQMQTAKTQPREEQFAGTYRVVSVLPIGHQDWLKGKQFTCKVNNKALPSPIERTISKARGQAHQPSVYVLPPSREELSKNTVSLTCLIKDFFPPDIDVEWQSNGQQEPESKYRTTPPQLDEDGSYFLYSKLSVDKSRWQRGDTFICAVMHEALHNHYTQESLSHSPGKCANINZED 4H3 (vL1) SEQ ID NO: 169GACATCGTGATGACCCAGACCCCTCTGAGCCTGTCCGTGAGCCCTGGCGAACCTGCCAGCATCAGCTGCAAGAGCAGCCAGAGCCTGCTGAACAGCAGGACCAGGAAGAACTACCTGGCCTGGTTCAGACAGAAGCCCGGCCAGAGCCCCCAGAGACTGATCTACTGGGCCAGCACCAGAGAGAGCGGCGTGCCTGACAGATTTAGCGGCAGCGGCAGCGGCACAGACTTCACCCTGAGGATCAGCAGAGTGGAGGCCGACGATGCCGGCGTGTACTACTGCAAGCAGAGCTACAACCTGTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGAGGAACGACGCTCAGCCAGCCGTGTACCTCTTCCAGCCTTCGCCGGACCAGCTTCATACGGGGTCAGCGTCGGTGGTGTGCCTGTTGAACTCGTTTTACCCCAAGGACATTAACGTGAAGTGGAAGGTAGACGGGGTAATTCAAGACACTGGCATTCAAGAGTCCGTCACGGAACAAGACTCAAAAGACTCAACGTATTCACTGTCGTCAACCTTGACGATGTCAAGCACCGAGTATCTTAGCCATGAGCTGTATTCGTGCGAGATCACCCACAAGTCCCTCCCCTCCACTCTTATCAAATCCTTTCAGCGGTCGGAATGTCAGCGGGTCGAT SEQ ID NO: 170DIVMTQTPLSLSVSPGEPASISCKSSQSLLNSRTRKNYLAWFRQKPGQSPQRLIYWASTRESGVPDRFSGSGSGTDFTLRISRVEADDAGVYYCKQSYNLYTFGQGTKVEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDSKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVDCANINZED 4H3 (vL2) SEQ ID NO: 171GACATCGTGATGACCCAGACCCCTCTGAGCCTGAGCGTGAGCCCTGGAGAGCCTGCCAGCATCAGCTGCAAGAGCAGCCAGAGCCTGCTGAACAGCAGGACCAGGAAGAACTACCTGGCCTGGTACAGGCAGAAGCCTGGCCAGAGCCCCCAGCTGCTGATCTACTGGGCCAGCACCAGAGAGAGCGGAGTGCCTGACAGGTTCAGCGGAAGCGGCAGCGGCACCGACTTCACCCTGAGGATCAGCAGAGTGGAGGCCGATGACGCCGGCGTGTACTACTGCAAGCAGAGCTACAACCTGTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGAGGAACGACGCTCAGCCAGCCGTGTACCTCTTCCAGCCTTCGCCGGACCAGCTTCATACGGGGTCAGCGTCGGTGGTGTGCCTGTTGAACTCGTTTTACCCCAAGGACATTAACGTGAAGTGGAAGGTAGACGGGGTAATTCAAGACACTGGCATTCAAGAGTCCGTCACGGAACAAGACTCAAAAGACTCAACGTATTCACTGTCGTCAACCTTGACGATGTCAAGCACCGAGTATCTTAGCCATGAGCTGTATTCGTGCGAGATCACCCACAAGTCCCTCCCCTCCACTCTTATCAAATCCTTTCAGCGGTCGGAATGTCAGCGGGTCGAT SEQ ID NO: 172DIVMTQTPLSLSVSPGEPASISCKSSQSLLNSRTRKNYLAWYRQKPGQSPQLLIYWASTRESGVPDRFSGSGSGTDFTLRISRVEADDAGVYYCKQSYNLYTFGQGTKVEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDSKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVDCANINZED 4H3 (vL3) SEQ ID NO: 173GACATCGTGATGACCCAGACCCCTCTGAGCCTGAGCGTGAGCCCTGGAGAGCCTGCCAGCATCAGCTGCAAGAGCAGCCAGAGCCTGCTGAACAGCAGGACCAGGAAGAACTACCTGGCCTGGTACCAGCAGAAGCCTGGCCAGAGCCCCCAGCTGCTGATCTACTGGGCCAGCACCAGAGAGAGCGGAGTGCCTGACAGGTTCAGCGGAAGCGGCAGCGGCACCGACTTCACCCTGAGGATCAGCAGAGTGGAGGCCGATGACGCCGGCGTGTACTACTGCAAGCAGAGCTACAACCTGTACACCTTCGGCCAGGGCACCAAGGTGGAGATCAAGAGGAACGACGCTCAGCCAGCCGTGTACCTCTTCCAGCCTTCGCCGGACCAGCTTCATACGGGGTCAGCGTCGGTGGTGTGCCTGTTGAACTCGTTTTACCCCAAGGACATTAACGTGAAGTGGAAGGTAGACGGGGTAATTCAAGACACTGGCATTCAAGAGTCCGTCACGGAACAAGACTCAAAAGACTCAACGTATTCACTGTCGTCAACCTTGACGATGTCAAGCACCGAGTATCTTAGCCATGAGCTGTATTCGTGCGAGATCACCCACAAGTCCCTCCCCTCCACTCTTATCAAATCCTTTCAGCGGTCGGAATGTCAGCGGGTCGAT SEQ ID NO: 174DIVMTQTPLSLSVSPGEPASISCKSSQSLLNSRTRKNYLAWYQQKPGQSPQLLIYWASTRESGVPDRFSGSGSGTDFTLRISRVEADDAGVYYCKQSYNLYTFGQGTKVEIKRNDAQPAVYLFQPSPDQLHTGSASVVCLLNSFYPKDINVKWKVDGVIQDTGIQESVTEQDSKDSTYSLSSTLTMSSTEYLSHELYSCEITHKSLPSTLIKSFQRSECQRVD

Example 8 Reactivity of Caninized Antibodies Against Canine IL-4Receptor Alpha

The caninized antibodies were tested for reactivity with canine IL-4receptor alpha as follows:

1. Coat 200 ng/well of IL-4 receptor alpha on an immunoplate andincubate the plate at 4° C. overnight.

2. Wash the plate 3 times with phosphate buffered saline (PBS)containing 0.05% Tween 20 (PBST).

3. Block the plate with 0.5% bovine serum albumin (BSA) in PBS for 45-60min at room temperature.

4. Wash the plate 3 times with PBST.

5. Three-fold dilute the caninized antibody in each column or row ofdilution plate starting at 0.3 μg/mL.

6. Transfer the diluted caninized antibody into each column or row ofthe immunoplate, and incubate the plate for 45-60 min at roomtemperature.

7. Wash the plate 3 times with PBST.

8. Add 1:4000 diluted horseradish peroxidase labeled anti-canine IgG Fcinto each well of the plate, and then incubate the plate for 45-60 minat room temperature.

9. Wash the plate 3 times with PBST.

10. Add 3,3′,5,5′-tetramethylbenzidine (TMB) Substrate into each well ofthe plate, and incubate the plate for 10 to 15 min at room temperatureto develop the color.

11. Add 100 μL 1.5 M phosphoric acid into each well to stop thereaction. Read plate at 450 nm with 540 nm reference wavelength.

As depicted in FIG. 4, the binding of five (5) antibodies to the IL-4receptor alpha was studied: 4H3 M-C, c4H3 H1-L1, c4H3 H2-L2, c4H3 H3-L3,and 2G9 M-C. 2G9 M-C was used as a negative control antibody. 4H3 M-C isa chimeric antibody consisting of the mouse variable heavy regions ofthe presently disclosed 4H3 antibody together with canine constantregions, and the light chain from the mouse 4H3 antibody. c4H3 H1-L1,c4H3 H2-L2, c4H3 H3-L3 are three caninized variants of the mouse 4H3antibody, and include specific heavy chains and light chains as depictedin Table 5 above. 2G9 M-C is a chimeric antibody consisting of the mousevariable heavy regions of a mouse antibody to an antigen that iscompletely unrelated to the IL-4 receptor alpha together with canineconstant regions, and the light chain from the mouse antibody to thatunrelated antigen. Consistently, 2G9 M-C did not bind to the IL-4receptor alpha, whereas the remaining four antibodies studied, i.e., 4H3M-C, c4H3 H1-L1, c4H3 H2-L2, and c4H3 H3-L3, all bound relativelytightly (see, FIG. 4).

1-33. (canceled)
 34. An isolated mammalian antibody or antigen bindingfragment thereof that binds canine interleukin-4 receptor α (IL-4R_(α))with specificity comprising three light chain complementary determiningregions (CDRs): a CDR light 1 (CDRL1), a CDR light 2 (CDRL2), and a CDRlight 3 (CDRL3); and three heavy chain CDRs: a CDR heavy 1 (CDRH1), aCDR heavy 2 (CDRH2), and a CDR heavy 3 (CDRH3); (a) wherein the CDRL1comprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 131, SEQ ID NO: 47, SEQ ID NO: 54, SEQ ID NO: 48, SEQ ID NO:52, SEQ ID NO: 55, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ IDNO: 53, SEQ ID NO: 129, or SEQ ID NO: 130, a conservatively modifiedvariant of SEQ ID NO: 131, SEQ ID NO: 47, SEQ ID NO: 54, SEQ ID NO: 48,SEQ ID NO: 52, SEQ ID NO: 55, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO:51, SEQ ID NO: 53, SEQ ID NO: 129, or SEQ ID NO: 130, a variant of SEQID NO: 47 or SEQ ID NO: 54 that comprises the canonical structure classof 1; a variant of SEQ ID NO: 48, SEQ ID NO: 52, or SEQ ID NO: 55 thatcomprises the canonical structure class of 2A; a variant of SEQ ID NO:49 that comprises the canonical structure class of 4; a variant of SEQID NO: 131, SEQ ID NO: 50, or SEQ ID NO: 51 that comprises the canonicalstructure class of 3; and a variant of SEQ ID NO: 53, SEQ ID NO: 129, orSEQ ID NO: 130 that comprises the canonical structure class of 6; (b)wherein the CDRL2 comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO: 60, SEQ ID NO: 56, SEQ ID NO: 57, SEQ IDNO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQID NO: 64, SEQ ID NO: 132, SEQ ID NO: 133, or SEQ ID NO: 134, aconservatively modified variant of SEQ ID NO: 60, SEQ ID NO: 56, SEQ IDNO: 57, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQID NO: 63, SEQ ID NO: 64, SEQ ID NO: 132, SEQ ID NO: 133, or SEQ ID NO:134, and a variant of SEQ ID NO: 60, SEQ ID NO: 56, SEQ ID NO: 57, SEQID NO: 58, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63,SEQ ID NO: 64, SEQ ID NO: 132, SEQ ID NO: 133, or SEQ ID NO: 134,wherein the variant comprises the canonical structure class of 1; (c)wherein the CDRL3 comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO: 138, SEQ ID NO: 65, SEQ ID NO: 66, SEQ IDNO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQID NO: 72, SEQ ID NO: 73, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO:137, or SEQ ID NO: 139, a conservatively modified variant of SEQ ID NO:138, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ IDNO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, or SEQ ID NO: 139, a variantof SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ IDNO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, or SEQ ID NO: 139 thatcomprises the canonical structure class of 1; and a variant of SEQ IDNO: 138 that comprises the canonical structure class of 3; (d) whereinthe CDRH1 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 142, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO:76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ IDNO: 81, SEQ ID NO: 82, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO:143, a conservatively modified variant of SEQ ID NO: 142, SEQ ID NO: 74,SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO:79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 140, SEQ IDNO: 141, or SEQ ID NO: 143, and a variant of SEQ ID NO: 142, SEQ ID NO:74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ IDNO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 140, SEQID NO: 141, or SEQ ID NO: 143, wherein the variant comprises thecanonical structure class of 1; (e) wherein the CDRH2 comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 147, SEQID NO: 83, SEQ ID NO: 84, SEQ ID NO: 91, SEQ ID NO: 85, SEQ ID NO: 88,SEQ ID NO: 89, SEQ ID NO: 87, SEQ ID NO: 90, SEQ ID NO: 144, SEQ ID NO:145, SEQ ID NO: 146, or SEQ ID NO: 148, a conservatively modifiedvariant of SEQ ID NO: 147, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 91,SEQ ID NO: 85, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 87, SEQ ID NO:90, SEQ ID NO: 144, SEQ ID NO: 145, SEQ ID NO: 146, or SEQ ID NO: 148, avariant of SEQ ID NO: 147, SEQ ID NO: 83, SEQ ID NO: 144, SEQ ID NO:146, or SEQ ID NO: 148 that comprises the canonical structure class of3A; a variant of SEQ ID NO: 84, SEQ ID NO: 91, or SEQ ID NO: 145 thatcomprises the canonical structure class of 2A; a variant of SEQ ID NO:85, SEQ ID NO: 88, or SEQ ID NO: 89 that comprises the canonicalstructure class of 2B; and a variant of SEQ ID NO: 87 or SEQ ID NO: 90that comprises the canonical structure class of 1; (f) wherein the CDRH3comprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 152, a conservatively modified variant of SEQ ID NO: 152, anda variant of SEQ ID NO: 152 that comprises the canonical structure classof 9; SEQ ID NO: 92, a conservatively modified variant of SEQ ID NO: 92,a variant of SEQ ID NO: 92 that comprises the canonical structure classof 12; SEQ ID NO: 93, a conservatively modified variant of SEQ ID NO:93, a variant of SEQ ID NO: 93 that comprises the canonical structureclass of 7; SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 149, or SEQ ID NO:151, a conservatively modified variant of SEQ ID NO: 94, SEQ ID NO: 96,SEQ ID NO: 149, or SEQ ID NO: 151, a variant of SEQ ID NO: 94, SEQ IDNO: 96, SEQ ID NO: 149, or SEQ ID NO: 151 that comprises the canonicalstructure class of 15; SEQ ID NO: 95, a conservatively modified variantof SEQ ID NO: 95, or a variant of SEQ ID NO: 95 that comprises thecanonical structure class of 11; SEQ ID NO: 97 or SEQ ID NO: 100, aconservatively modified variant of SEQ ID NO: 97 or SEQ ID NO: 100, avariant of SEQ ID NO: 97 or SEQ ID NO: 100 that comprises the canonicalstructure class of 6; SEQ ID NO: 98, a conservatively modified variantof SEQ ID NO: 98, a variant of SEQ ID NO: 98 that comprises thecanonical structure class of 4; SEQ ID NO: 99 or SEQ ID NO: 153, aconservatively modified variant of SEQ ID NO: 99 or SEQ ID NO: 153, avariant of SEQ ID NO: 99 or SEQ ID NO: 153 that comprises the canonicalstructure class of 13; and SEQ ID NO: 150, a conservatively modifiedvariant of SEQ ID NO: 150, a variant of SEQ ID NO: 150 that comprisesthe canonical structure class of 10; wherein the antibody and antigenbinding fragment thereof bind canine IL-4R_(α).
 35. The isolatedmammalian antibody or antigen binding fragment of claim 34, (a) whereinthe CDRL1 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 131, SEQ ID NO: 54, SEQ ID NO: 129, or SEQ IDNO: 130, a conservatively modified variant of SEQ ID NO: 131, SEQ ID NO:54, SEQ ID NO: 129, or SEQ ID NO: 130, a variant of SEQ ID NO: 54 thatcomprises the canonical structure class of 1, a variant of SEQ ID NO:131 that comprises the canonical structure class of 3, and a variant ofSEQ ID NO: 129 or SEQ ID NO: 130 that comprises the canonical structureclass of 6; (b) wherein the CDRL2 comprises an amino acid sequenceselected from the group consisting of SEQ ID NO: 60, SEQ ID NO: 63, SEQID NO: 132, SEQ ID NO: 133, SEQ ID NO: 134, a conservatively modifiedvariant of SEQ ID NO: 60, SEQ ID NO: 63, SEQ ID NO: 132, SEQ ID NO: 133,or SEQ ID NO: 134, and a variant of SEQ ID NO: 60, SEQ ID NO: 63, SEQ IDNO: 132, SEQ ID NO: 133, or SEQ ID NO: 134, wherein the variantcomprises the canonical structure class of 1; (c) wherein the CDRL3comprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 138, SEQ ID NO: 72, SEQ ID NO: 135, SEQ ID NO: 136, SEQ IDNO: 137, SEQ ID NO: 139, a conservatively modified variant of SEQ ID NO:138, SEQ ID NO: 72, SEQ ID NO: 135, SEQ ID NO: 136, SEQ ID NO: 137, orSEQ ID NO: 139, a variant of SEQ ID NO: 72, SEQ ID NO: 135, SEQ ID NO:136, SEQ ID NO: 137, or SEQ ID NO: 139 that comprises the canonicalstructure class of 1; and a variant of SEQ ID NO: 138 that comprises thecanonical structure class of 3; (d) wherein the CDRH1 comprises an aminoacid sequence selected from the group consisting of SEQ ID NO: 142, SEQID NO: 81, SEQ ID NO: 140, SEQ ID NO: 141, SEQ ID NO: 143, aconservatively modified variant of SEQ ID NO: 142, SEQ ID NO: 81, SEQ IDNO: 140, SEQ ID NO: 141, or SEQ ID NO: 143, and a variant of SEQ ID NO:142, SEQ ID NO: 81, SEQ ID NO: 140, SEQ ID NO: 141, or SEQ ID NO: 143,wherein the variant comprises the canonical structure class of 1; (e)wherein the CDRH2 comprises an amino acid sequence selected from thegroup consisting of SEQ ID NO: 147, SEQ ID NO: 90, SEQ ID NO: 144, SEQID NO: 145, SEQ ID NO: 146, SEQ ID NO: 148, a conservatively modifiedvariant of SEQ ID NO: 147, SEQ ID NO: 90, SEQ ID NO: 144, SEQ ID NO:145, SEQ ID NO: 146, SEQ ID NO: 148, a variant of SEQ ID NO: 147, SEQ IDNO: 144, SEQ ID NO: 146, or SEQ ID NO: 148 that comprises the canonicalstructure class of 3A; a variant of SEQ ID NO: 145 that comprises thecanonical structure class of 2A; and a variant of SEQ ID NO: 90 thatcomprises the canonical structure class of 1; (f) wherein the CDRH3comprises an amino acid sequence selected from the group consisting ofSEQ ID NO: 149, SEQ ID NO: 151, a conservatively modified variant of SEQID NO: 149 or SEQ ID NO: 151, a variant of SEQ ID NO: 149 or SEQ ID NO:151 that comprises the canonical structure class of 15, SEQ ID NO: 99,SEQ ID NO: 153, a conservatively modified variant of SEQ ID NO: 99 orSEQ ID NO: 153, a variant of SEQ ID NO: 99 or SEQ ID NO: 153 thatcomprises the canonical structure class of 13, SEQ ID NO: 150, aconservatively modified variant of SEQ ID NO: 150, a variant of SEQ IDNO: 150 that comprises the canonical structure class of 10, SEQ ID NO:152, a conservatively modified variant of SEQ ID NO: 152, and a variantof SEQ ID NO: 152 that comprises the canonical structure class of 9;wherein the antibody and antigen binding fragment thereof bind canineIL-4R_(α) and block the binding of canine IL-4R_(α) to canineinterleukin-4.
 36. The isolated mammalian antibody or antigen bindingfragment of claim 35, (a) wherein the CDRL1 comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 54, aconservatively modified variant of SEQ ID NO: 54, and a variant of SEQID NO: 54 that comprises the canonical structure class of 1; (b) whereinthe CDRL2 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 63, a conservatively modified variant of SEQ IDNO: 63, and a variant of SEQ ID NO: 63 that comprises the canonicalstructure class of 1; (c) wherein the CDRL3 comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 72, aconservatively modified variant of SEQ ID NO: 72, and a variant of SEQID NO: 72 that comprises the canonical structure class of 1; (d) whereinthe CDRH1 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 81, a conservatively modified variant of SEQ IDNO: 81, and a variant of SEQ ID NO: 81 that comprises the canonicalstructure class of 1; (e) wherein the CDRH2 comprises an amino acidsequence selected from the group consisting of SEQ ID NO: 90, aconservatively modified variant of SEQ ID NO: 90, and a variant of SEQID NO: 90 that comprises the canonical structure class of 1; (f) whereinthe CDRH3 comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 99, a conservatively modified variant of SEQ IDNO: 99, and a variant of SEQ ID NO: 99 that comprises the canonicalstructure class of 13; and wherein the antibody and antigen bindingfragment thereof bind canine IL-4R_(α) and block the binding of canineIL-4R_(α) to canine interleukin-4.
 37. The isolated mammalian antibodyor antigen binding fragment thereof of claim 34 that is a caninizedantibody or a caninized antigen binding fragment thereof.
 38. Thecaninized antibody of claim 37 or caninized antigen binding fragmentthereof, that comprises a hinge region that comprises the amino acidsequence selected from the group consisting of SEQ ID NO: 101, SEQ IDNO: 102, SEQ ID NO: 103, and SEQ ID NO:
 104. 39. The isolated caninizedantibody or antigen binding fragment thereof of claim 36, that is acaninized antibody or a caninized antigen binding fragment thereof. 40.The isolated caninized antibody or antigen binding fragment thereof ofclaim 39, wherein when bound to canine IL-4R_(α), said caninizedantibody or antigen binding fragment thereof binds to at least one aminoacid residue within the amino acid sequence of SEQ ID NO: 125, SEQ IDNO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 154, SEQ ID NO: 155,SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, SEQ ID NO: 159, SEQ IDNO: 160, SEQ ID NO: 161, SEQ ID NO: 162 or any combination thereof;wherein the antibody or antigen binding fragment thereof binds canineIL-4R_(α) and blocks the binding of canine IL-4R_(α) to canineinterleukin-4.
 41. A caninized monoclonal antibody or antigen bindingfragment thereof that cross-competes for binding with canine IL-4R_(α)with the caninized antibody; wherein the caninized monoclonal antibodyand antigen binding fragment thereof bind canine IL-4R_(α) and block thebinding of canine IL-4R_(α) to canine IL-4.
 42. An isolated nucleic acidcomprising a nucleotide sequence encoding a protein selected from thegroup consisting of a light chain of the caninized antibody of claim 37and an antigen binding fragment said light chain.
 43. An expressionvector comprising the isolated nucleic acid of claim
 42. 44. A host cellcomprising the expression vector of claim
 43. 45. An isolated nucleicacid comprising a nucleotide sequence encoding a protein selected fromthe group consisting of a heavy chain of the caninized antibody of claim37, and an antigen binding fragment said heavy chain.
 46. An expressionvector comprising the isolated nucleic acid of claim
 45. 47. A host cellcomprising the expression vector of claim
 46. 48. A pharmaceuticalcomposition comprising the caninized antibody of claim 37 and apharmaceutically acceptable carrier or diluent.
 49. A pharmaceuticalcomposition comprising the caninized antibody of claim 39 and apharmaceutically acceptable carrier or diluent.
 50. A method ofdecreasing the activity of an immune cell, comprising administering to asubject in need thereof a therapeutically effective amount of thepharmaceutical composition of claim
 49. 51. The method of claim 50,wherein said method is used for: the treatment of atopic dermatitis; or(ii) the treatment of asthma; or (iii) the treatment of atopicdermatitis and the treatment of asthma.
 52. A method of decreasing theactivity of an immune cell, comprising administering to a subject inneed thereof a therapeutically effective amount of the pharmaceuticalcomposition of claim 48; wherein said method is used as a treatmentselected from the group consisting of atopic dermatitis, asthma, andboth atopic dermatitis and asthma.